Monospecific and bispecific anti-igf-1r and anti-erbb3 antibodies

ABSTRACT

Provided are monospecific and bispecific antibodies that are useful as anti-neoplastic agents and that bind specifically to human IGF-1R and human ErbB3. Exemplary antibodies inhibit signal transduction through either or both of IGF-1R and ErbB3. Exemplary polyvalent proteins comprise at least one anti-IGF-1R binding site and at least one anti-ErbB3 binding site. In certain embodiments the binding sites may be linked through an immunoglobulin constant region. AntiErbB3 and anti-IGF-1R antibodies (e.g., monoclonal antibodies) are also provided.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/451,135, filed Apr. 19, 2012 which claims priority to U.S.Provisional Application No. 61/477,089, filed Apr. 19, 2011; U.S.Provisional Application No. 61/539,297, filed Sep. 26, 2011; U.S.Provisional Application No. 61/558,192, filed Nov. 10, 2011 and U.S.Provisional Application No. 61/619,244, filed Apr. 2, 2012. Wherepermitted, the foregoing applications are incorporated by reference,each in its entirety, for any and all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 5, 2012, isnamed 1141PC01.txt and is 950,611 bytes in size.

BACKGROUND

Tumor cells express receptors for growth factors and cytokines thatstimulate proliferation of the cells. Antibodies to such receptors canbe effective in blocking the stimulation of cell proliferation mediatedby growth factors and cytokines and can thereby inhibit tumor cellproliferation and tumor growth. Commercially available therapeuticantibodies that target receptors on cancer cells include, for example,trastuzumab which targets the HER2 receptor (also known as ErbB2) forthe treatment of breast cancer, and cetuximab which targets theepidermal growth factor receptor (EGFR, also known as HER1 or ErbB1) forthe treatment of colorectal cancer and head and neck cancer.

Monoclonal antibodies have significantly advanced our ability to treatcancers, yet clinical studies have shown that many patients do notadequately respond to monospecific therapy. This is in part due to themultigenic nature of cancers, where cancer cells rely on multiple andoften redundant pathways for proliferation. Bi- or multi-specificantibodies capable of blocking multiple growth and survival pathways atonce have a potential to better meet the challenge of blocking cancergrowth, and indeed many of them are advancing in clinical development.However, bispecific antibodies present significant design challenges,due to the greatly increased number of variables that need to beconsidered in their design and optimization, as well as to theirstructural differences from naturally occurring antibodies.

Monoclonal antibodies such as trastuzumab, cetuximab, bevacizumab andpanitumumab have significantly improved patient outcomes in the clinic,and over two hundred therapeutic monoclonal antibodies are currentlybeing tested in clinical development. However, it has become apparentthat tumors driven by single oncogenes are not the norm, and treatmentoften results in activation of resistance mechanisms, which in turn alsorequire targeted intervention. For example, in multiple pre-clinicalmodels of trastuzumab resistance, inhibition of IGF-1R restoressensitivity to trastuzumab. Combination of targeted agents has beenattempted in the clinic, but so far they have had limited clinicalsuccess and in combination can be prohibitively expensive. The need toinhibit multiple targets either due to resistance or to tumors beingdriven by multiple growth factor pathways has led to increased interestin bispecific antibodies. Currently developed bispecific antibodies weretypically designed in empirical fashion. Further, the pharmaceuticalproperties of these bispecific antibodies were almost invariablyinferior to those of monoclonal antibodies. These factors present amajor challenge to the development of bispecific anti-cancer therapies.Significant added benefit from targeting multiple cancer survivalpathways can be derived from increased work up front to identify andengineer a bispecific antibody with optimal characteristics. Thisrequires an iterative approach consisting of computational simulation toidentify optimal targeting strategies and design specifications,engineering of inhibitors that possess these characteristics, andexperimental validation of the therapeutic hypothesis. We separate thisengineering framework into two categories: selection of appropriatemolecular format with robust pharmaceutical properties and computationalsimulation to identify the best targets and optimal therapeutic designcharacteristics, e.g., in an IgG-like bispecific antibody (FIG. 8).

One of the main advantages of antibodies is their ability to bindtightly to virtually any extracellular target. This property is drivenby two features of antibody variable regions (VRs): the large flatsurface of six complementarity determining regions (CDRs) and the factthat antibodies have two binding arms that can target two moleculessimultaneously. In bispecific antibodies, dual target binding results intighter affinity because once one arm of the antibody is bound to anextracellular target, the second arm is restricted to a narrow regionabove the plasma membrane (about 100 angstroms), and is, therefore,concentrated near the cell surface. This results in a much fastersecondary binding event that is not limited by diffusion. Theacceleration of a secondary binding event is called. Both affinity andavidity are rationally engineerable properties, as the former can beimproved via in silico affinity maturation and the latter can beenhanced by engineering additional targeting arms to the same ordifferent antigen present on the cell surface. In addition to theirbinding capabilities, antibodies may possess multiple effector functionsmediated by their Fc domain: antibody-dependent cellular cytotoxicity(ADCC) and antibody-dependent cellular phagocytosis (ADCP) that inhumans are determined by interactions with activating FcγRI, FcγRIIa/c,FcγRIIIa and inhibitory FcγRIIb receptors; complement-dependentcytotoxicity (CDC) that is triggered by antibody binding to thecomponents of the complement system; and long half-life that is mediatedvia active recycling by the neonatal Fc receptor (FcRn). All of thesefunctions can be tuned to optimize the effectiveness of an anti-cancertherapy and are may be retained to advantage in a bispecific protein.

The variable fragment (Fv), composed of the variable heavy (VH) andvariable light (VL) domains of an IgG antibody, is a minimal antibodyfragment that displays full antigen binding. These variable domains canbe successfully fused into a single chain construct (scFv), althoughaffinity is often reduced to some extent compared to a whole nativeantibody. A majority of current bispecific formats feature one orseveral scFv modules attached to the N- or C-terminus of an IgG heavychain or IgG light chain via a low complexity linker Another bispecificantibody format is dual variable domain immunoglobulin (DVD-Ig). DVD-Igconsists of a first IgG heavy chain with a second VH domain linked toits N-terminus by a short linker, and a first IgG light chain with asecond VL domain similarly linked to its N-terminus. The second VH/VLdomains form a pair with specificity for one antigen while the firstVH/VL form a separate binding site with specificity for a differentantigen.

Bivalent formats of IgG-like antibodies have one potential limitation;they can cross-link cell surface antigens, some of which are activatedby dimerization, triggering undesirable signaling events in anuncontrolled manner. To address this challenge, tunable monovalentbispecific antibody formats have been developed. MetMab, a one armedanti-c-Met therapeutic antibody created by incorporating asymmetric“knobs and holes” into the Fc fragment, has been shown to be effectivein models of pancreatic cancer and is being investigated in multipleclinical trials. This “knobs and holes” format has been recentlyextended to incorporate an antibody fragment targeting EGFR, giving riseto a functionally monovalent bispecific protein targeting EGFR/ErbB1 andc-Met/HGFR. Gunasekaran et al. have described an alternativeimplementation of the “knobs and holes “concept by engineeringcomplementary charged surfaces into the Fc fragment. Davis et al. havedescribed a “SEED” approach that used modified asymmetric Fc containingfragments from human IgG and IgA to form heteromeric monovalentantibodies. Finally, Bostrom et al. has described a novel engineeringapproach to construct bifunctional Fab fragment that can bind eitherHER2 or VEGF with high affinity. When combined in a canonical antibodymolecule, these Fab fragments will engage HER2 or VEGF with differentvalences that will depend on the cellular environment and growth factorconcentration.

Another important component of a bispecific antibody design is theoptimization of pharmaceutical properties. To be clinically useful, atherapeutic protein must be stable, remain soluble over an extendedperiod of time and possess a robust manufacturability profile.Bispecific antibodies are typically less stable than monoclonalantibodies, and initially may not possess adequate pharmaceuticalproperties for development. They can be stabilized through molecularengineering, through downstream formulation activities, or, as mostcommonly practiced, through the combination of both approaches.

The importance of minimizing chemical manufacturing and controlliabilities in small molecule drug candidates has been long recognizedand the rules to predict drug-likeness have been proposed. Many groupshave used conceptually similar approaches to assess the fitness of IgGbased proteins by evaluating unfavorable sequence features such as:non-canonical disulfides or unpaired cysteines, extra glycosylationsites, tyrosine sulfation motifs, solvent accessible methionines,asparagine deamidation motifs, and acid cleavage sites. Extraglycosylation sites and asparagine deamidation sites are quite commonfeatures of natural antibodies sequences. In fact over 20% of variabledomains of heavy chains are reported to be glycosylated and over 5% ofgermline genes contain asparagine-glycine deamidation motifs.Deamidation rates in antibodies can be reliably estimated using themethod proposed by Robinson that suggests that structurally constrainedloops do not form a succinimide intermediate efficiently and thereforeare stable.

While the canonical N-linked glycosylation motif (NXS and NXT, where Xis any aa but proline) can be easily detected in antibody sequence,O-linked sites, which are liabilities in that they can also negativelyimpact pharmaceutical properties, are more difficult to recognize.Recently several O-linked modifications of variable domains of antibodylight chains have been reported, mostly in the proximity of GS richsequence motifs. Many approaches to improve the affinity and stabilityof a candidate protein at the discovery stage exist includingstructure-guided design, focused library screening and yeast display;therefore, we find it beneficial to remove such potential liabilities atrisk in the early proof-of-concept proteins.

Other liabilities, such as aggregation and immunogenicity are morechallenging from an engineering perspective. Not only are bothmultifaceted properties, but it is also very difficult to adequatelyevaluate them in small scale biochemical and biophysical testing and,therefore, they tend to first be detected late in development. Arguablythe best approach to reduce antibody immunogenicity is throughhumanization. This approach has been extensively validated by a numberof humanized antibodies that have been well-tolerated in the clinic. Arecently proposed “superhumanization” approach introduces human germlinesequences into the CDRs to yield ‘fully human’ antibodies. This approachrequires each amino acid (“aa”) in the CDRs to be mutated in order todetermine its contribution to antigen binding; the resulting antibodymay be less immunogenic. Aside from sequence-based features,immunogenicity of antibodies and antibody-like proteins can be dependenton their aggregation stability. Interestingly, the “humanness” and thestability in an antibody module, such as scFv, can be co-engineered viaa knowledge-based approach.

Engineering of protein antibody solubility is another daunting task, asthe property is also a composite of several physico-chemical parameters.Nevertheless, a number of methods to combat insolubility have beenproposed. Pepinsky et al. have used glycoengineering, isotype switching,and structure-guided mutagenesis, to increase the solubility of amonoclonal antibody. Chemamsetty et al. have described an unbiasedapproach to improving stability with respect to aggregation that relieson molecular dynamics simulations to calculate a parameter calledsurface aggregation propensity and applied this technique to introducestabilizing amino acids (“aas”) in the aggregation-prone regions ofantibodies. Interestingly, in their analysis of antibodies, aggregationprone regions often co-locate with functionally important regions thatconfer Fc receptor or antigen binding and, therefore, cannot be easilyremoved.

Such a coupling between molecular function and pharmaceutical propertiesin antibodies is common. It can significantly complicate optimization ofthe bispecific antibodies, as even larger parts of their sequences arelocated in the functionally important regions. Therefore, to enablesuccessful engineering it is important to identify critical molecularfunctions and optimal design characteristics.

Computational Simulation to Identify Best Targets and OptimalTherapeutic Design Characteristics

Computational simulation is a valuable tool for guiding drug developmentdecisions, both in the laboratory and in the clinic. Populationpharmacokinetic modeling is a mature example of the use of models tooptimize dose scheduling and clinical trial designs. For therapies withknown targets it is possible to utilize computational simulation muchearlier in the design process. Advances in multiplex, high-throughputquantitative protein measurement technologies have enabled observationof the complex dynamics that occur in cellular signaling networks. Thesedata permit the creation of network models which capture the mechanisticbehaviors of biological systems that are relevant to diseases such ascancer. By simulating potential therapeutics through network modeling itis possible to design more effective therapeutics at an accelerated paceand more accurately predict design parameters.

Traditionally, selection of a pharmaceutical agent for a targetedtherapy begins with a known target which is selected from a multitude ofmolecular, biological and physiological data. However, even inwell-studied and heavily targeted biological systems there areopportunities for new discoveries, which can be aided by simulation ofpathway or network models.

Accordingly, additional therapeutic approaches for cancer treatment, andin particular polyspecific antibody-based proteins that are engineeredto have superior biophysical and therapeutic properties are difficult toobtain, but are needed to overcome limitations of current antibodytherapies and to provide other benefits.

SUMMARY

Provided herein are polyvalent bispecific antibodies (PBA), whichantibodies are proteins comprising two pairs of polypeptide chains, eachpair of said two pairs comprising a heavy chain joined to a light chainby at least one heavy-light chain bond; wherein (a) each pair comprisesat least one anti-IGF-1R binding site and at least one anti-ErB3 bindingsite; and (b) each pair comprises a first binding site that comprises anN-terminal portion of the heavy chain of the PBA and an N-terminalportion of the light chain of the PBA, and a second binding site that isa C-terminal scFv that is entirely comprised by the heavy chain of thePBA, said C-terminal scFv containing a heavy chain VR joined to a lightchain VR by an scFv linker; and the anti-IGF-1R binding site is linkedto the anti-ErbB3 binding site through a heavy chain immunoglobulin (HCIg) constant region (CR) comprised by the heavy chain of the PBA, andthe two pairs are conjoined by at least one bond between the HC Ig CRsof each pair. In preferred embodiments, the anti-IGF-1R binding sitecomprises a heavy chain variable (VH) domain comprising a set of threeVH Complementarity Determining Regions (CDRs) comprising either (a)VHCDR1 (aa numbers 26-35), VHCDR2 (aa numbers 51-66), and VHCDR3 (aanumbers 99-111), of a heavy chain having an aa sequence comprising theaa sequence of (set forth in) a SEQ ID NO selected from the groupconsisting of SEQ ID NO:1, SEQ ID NOs:8-31 and SEQ ID NOs:384-385; or(b) a set of three VH Complementarity Determining Regions (CDRs)comprising VHCDR1 comprising SEQ ID NO:302, VHCDR2 comprising SEQ IDNO:303 and VHCDR3 comprising SEQ ID NO:304, and a light chain variable(VL) domain comprising a set of three VLCDRs comprising either (c)VLCDR1 (aa numbers 24-34), VLCDR2 (aa numbers 50-56) and VLCDR3 (aanumbers 89-97) of a light chain having an aa sequence comprising the aasequence of a SEQ ID NO selected from the group consisting of SEQ IDNOs:2-3, SEQ ID NOs:32-133, and SEQ ID NOs:386-387; or (d) a set ofthree VLCDRs comprising VLCDR1 comprising SEQ ID NO:305, VLCDR2comprising SEQ ID NO:306 and VLCDR3 comprising SEQ ID NO:307 or SEQ IDNO:308, and each CDR further comprising an amino terminus and a carboxyterminus, wherein the CDRs of each set of CDRs are arranged in thecorresponding heavy or light chain in a linear amino to carboxy order ofCDR1, CDR2 and CDR3, or wherein the sequences of VHCDR1, VHCDR2 andVHCDR3 comprise variable aas, which independently represent any aa setforth at the corresponding position in FIG. 1, and the sequences ofVLCDR1, VLCDR2 and VLCDR3 comprise variable aas, which independentlyrepresent any aa set forth at the corresponding position in FIG. 2, withthe proviso that the PBA (i) does not comprise both the anti-IGF-1R SFmodule and the anti-ErbB3 C8 module; or (ii) comprises at least one CDRor FR that differs in one or more aas from a CDR or FR, respectively, ofthe SF or C8 module. In certain embodiments, the anti-ErbB3 binding sitecomprises a VH domain comprising a set of three VH CDRs comprisingeither (e) VHCDR1 (aa numbers 26-35), VHCDR2 (aa numbers 51-66) andVHCDR3 (aa numbers 99-111) of a heavy chain having an aa sequencecomprising the aa sequence of a SEQ ID NO selected from the groupconsisting of SEQ ID NOs:4-5, SEQ ID NOs: 134-165, and SEQ ID NO:388, or(f) a set of three VH CDRs comprising VHCDR1 comprising SEQ ID NO:309,VHCDR2 comprising SEQ ID NO:310 and VHCDR3 comprising SEQ ID NO:311, anda light chain variable (VL) domain comprising a set of three VLCDRscomprising either (g) VLCDR1 (aa numbers 23-33), VLCDR2 (aa numbers49-55) and VLCDR3 (aa numbers 88-98), of a light chain having an aasequence comprising the aa sequence of a SEQ ID NO selected from thegroup consisting of SEQ ID NOs:6-7 and SEQ ID NOs: 166-200; or (h) alight chain variable (VL) domain comprising a set of three VLCDRscomprising VLCDR1 comprising SEQ ID NO:312, VLCDR2 comprising SEQ IDNO:313 and VLCDR3 comprising SEQ ID NO:314 or SEQ ID NO:315, and eachCDR further comprises an amino terminus and a carboxy terminus, whereinthe CDRs of each set of CDRs are arranged in the antibody in a linearamino to carboxy order of CDR1, CDR2 and CDR3, or wherein the sequencesof the VHCDR1, VHCDR2 and VHCDR3 comprise variable aas, whichindependently represent any aa set forth at the corresponding positionin FIG. 3, and the sequences of the VLCDR1, VLCDR2 and VLCDR3 comprisevariable aas, which independently represent any aa set forth at thecorresponding position in FIG. 4, wherein the PBA (i) does not compriseboth the anti-IGF-1R module comprising a light chain comprising SEQ IDNO:35 and a heavy chain comprising SEQ ID NO:11 and the b) an anti-ErbB3C8 module comprising a light chain comprising SEQ ID NO:175 and a heavychain comprising SEQ ID NO:145. In certain embodiments, the anti-IGF-1RVLCDR3 comprises SEQ ID NO:308 or the anti-ErbB3 VLCDR3 comprises SEQ IDNO:315. In certain embodiments, the two pairs of polypeptide chains arehave essentially identical sequences. At least one of at least one bondsbetween the HC Ig CRs bonds is a disulfide bond and may be a disulfidebond or a van der Waals bond or at least one of said at least oneheavy-light chain bonds is a disulfide bond and may be a disulfide bondor a van der Waals bond. In certain embodiments, the anti-ErbB3 bindingsite is the C-terminal scFv and in certain embodiments, the anti-IGF-1Rbinding site is the C-terminal scFv. The anti-IGF-1R binding site, theHC Ig CR and the anti-ErbB3 binding site of a PBA may comprise the heavychain of that pair, which is comprised by a single, contiguouspolypeptide chain.

A PBA may (i) inhibit growth of tumor cells in vitro at a concentrationof 1 μM or less, or 100 nM or less, or 10 nM or less, or 1 nM or less,or ii) inhibits either or both of heregulin and IGF1 induced signaltransduction with an IC50 of 10 nM or less or 1 nM or less or 100 pM orless, or a maximal percent inhibition of at least 70% or at least 80% orat least 90%, as indicated by inhibition of phosphorylation of either orboth of pErbB3 and pIGF-1R. Growth inhibition may be measured with a CTGassay in DU145 cells in culture. Inhibition of signal transduction maybe determined in BxPC-3 cells in culture following stimulation withIGF-1 at 80 ng/ml and heregulin at 20 ng/ml for 15 minutes.

In certain embodiments, each HC Ig CR of a PBA comprises a CH3 domainthat mediates conjunction with the CH3 domain of the other pair. Each HCIg CR may also comprise a CH2 domain, a hinge, and a CH1 domain. Incertain embodiments, the CH1 domain of a PBA is linked at its C-terminusto the N-terminus of a hinge, which is linked at its C-terminus to theN-terminus of a CH2 domain, which is linked at its C-terminus to theN-terminus of a CH3 domain.

Each first binding site may comprise a first VH domain, and each CH1domain of a PBA may be linked at its N-terminus to the C-terminus of thefirst VH domain. Each CH3 domain of a PBA may be linked at itsC-terminus to the N-terminus of the scFv. Each CH3 domain of a PBA maybe linked at its C terminus to the N-terminus of a connecting linker,which is linked at its C-terminus to the N-terminus of the scFv. Eachlight chain may comprise a first VL domain that associates with thefirst VH domain to form the first binding site. Each first VL domain maybe linked at its C-terminus to the N-terminus of a CL domain. Each firstbinding site may be an anti-IGF-1R binding site and each scFv may be ananti-ErbB3 scFv. Each first binding site may be an anti-ErbB3 bindingsite and each scFv may be an anti-IGF-1R scFv. The HC Ig CR of a PBA maybe an IgG CR, e.g., an IgG1 or IgG2 CR.

In certain embodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBAcomprise the aa sequence of the corresponding CDRs of SEQ ID NO:8 andthe anti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:32. In certainembodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:9 and theanti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:33. In certainembodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:10 and theanti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:34. In certainembodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:11 and theanti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:35. In certainembodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:8 and theanti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:33. In certainembodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:10 and theanti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:32.

In certain embodiments, the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBAcomprise the aa sequence of the corresponding CDRs of SEQ ID NO:134 andthe anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:166. In certainembodiments, the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBA comprise theaa sequence of the corresponding CDRs of SEQ ID NO:135 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NO:167. In certain embodiments, theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:136 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:168. In certain embodiments, theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:137 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:169. In certain embodiments, theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:138 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:170. In certain embodiments, theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:139 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:171. In certain embodiments, theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:140 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:172. In certain embodiments, theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:141 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NOs: 173. In certain embodiments, theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:142 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:174. In certain embodiments, theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:143 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:175. In certain embodiments, theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of a PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:136 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:169.

In certain embodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBAcomprise the aa sequence of the corresponding CDRs of SEQ ID NO:8 andthe anti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:32; and (a) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:134 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:166; or (b) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:135 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:167; or (c) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:136 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NO:168; or (d) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:137 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:169; or (e) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:138and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:170; or (f) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:139 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:171; or (g) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:140 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:172; or (h) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:141 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NOs: 173; or (i) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:142 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:174; or (j) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:143and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:175; or (k) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:136 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:169.

In certain embodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBAcomprise the aa sequence of the corresponding CDRs of SEQ ID NO:9 andthe anti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:33; and (a) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:134 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:166; or (b) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:135 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:167; or (c) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:136 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NO:168; or (d) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:137 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:169; or (e) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:138and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:170; or (f) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:139 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:171; or (g) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:140 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:172; or (h) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:141 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NOs: 173; or (i) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:142 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:174; or (j) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:143and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:175; or (k) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:136 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:169.

In certain embodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBAcomprise the aa sequence of the corresponding CDRs of SEQ ID NO:10 andthe anti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:34; and (a) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:134 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:166; or (b) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:135 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:167; or (c) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:136 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NO:168; or (d) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:137 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:169; or (e) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:138and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:170; or (f) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:139 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:171; or (g) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:140 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:172; or (h) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:141 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NOs: 173; or (i) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:142 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:174; or (j) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:143and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:175; or (k) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:136 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:169.

In certain embodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBAcomprise the aa sequence of the corresponding CDRs of SEQ ID NO:11 andthe anti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:35; and (a) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:134 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:166; or (b) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:135 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:167; or (c) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:136 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NO:168; or (d) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:137 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:169; or (e) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:138and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:170; or (f) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:139 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:171; or (g) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:140 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:172; or (h) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:141 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NOs: 173; or (i) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:142 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:174; or (j) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:136and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:169.

In certain embodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBAcomprise the aa sequence of the corresponding CDRs of SEQ ID NO:8 andthe anti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:33; and (a) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:134 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:166; or (b) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:135 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:167; or (c) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:136 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NO:168; or (d) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:137 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:169; or (e) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:138and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:170; or (f) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:139 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:171; or (g) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:140 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:172; or (h) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:141 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NOs: 173; or (i) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:142 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:174; or (j) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:143and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:175; or (k) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:136 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:169.

In certain embodiments, the anti-IGF-1R VHCDR1, VHCDR2, VHCDR3 of a PBAcomprise the aa sequence of the corresponding CDRs of SEQ ID NO:10 andthe anti-IGF-1R VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:32; and (a) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:134 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:166; or (b) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:135 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:167; or (c) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:136 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NO:168; or (d) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:137 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:169; or (e) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:138and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:170; or (f) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:139 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:171; or (g) the anti-ErbB3 VHCDR1,VHCDR2, VHCDR3 of the PBA comprise the aa sequence of the correspondingCDRs of SEQ ID NO:140 and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 ofthe PBA comprise the aa sequence of the corresponding CDRs of SEQ IDNO:172; or (h) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprisethe aa sequence of the corresponding CDRs of SEQ ID NO:141 and theanti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aa sequenceof the corresponding CDRs of SEQ ID NOs: 173; or (i) the anti-ErbB3VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:142 and the anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 of the PBA comprise the aa sequence of the corresponding CDRsof SEQ ID NO:174; or (j) the anti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of thePBA comprise the aa sequence of the corresponding CDRs of SEQ ID NO:143and the anti-ErbB3 VLCDR1, VLCDR2 and VLCDR3 of the PBA comprise the aasequence of the corresponding CDRs of SEQ ID NO:175; or (k) theanti-ErbB3 VHCDR1, VHCDR2, VHCDR3 of the PBA comprise the aa sequence ofthe corresponding CDRs of SEQ ID NO:136 and the anti-ErbB3 VLCDR1,VLCDR2 and VLCDR3 of the PBA comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:169.

In certain embodiments, each anti-IGF-1R binding site of a PBA comprisesa VH domain comprising the sequence of SEQ ID NO:1, wherein the sequencecomprises variable aas, which independently represent any aa set forthat the corresponding position in FIG. 1 and/or each anti-IGF-1R bindingsite of a PBA comprises a VL domain comprising the sequence of SEQ IDNO:2 (or 3), wherein the sequence comprises variable aas, whichindependently represent any aa set forth at the corresponding positionin FIG. 2.

In certain embodiments, each anti-ErbB3 binding site of a PBA comprisesa VH domain comprising the sequence of SEQ ID NO:4 (or 5), wherein thesequence comprises variable aas, which independently represent any aaset forth at the corresponding position in FIG. 3 and/or each anti-ErbB3binding site of a PBA comprises a VL domain comprising the sequence ofSEQ ID NO:6 (or 7), wherein the sequence comprises variable aas, whichindependently represent any aa set forth at the corresponding positionin FIG. 4.

In certain embodiments, each anti-IGF-1R binding site of a PBA comprisesa VH domain comprising the sequence of SEQ ID NO:1 and a VL domaincomprising the sequence of SEQ ID NO:2 (or 3) and each anti-ErbB3binding site of the PBA comprises a VH domain comprising the sequence ofSEQ ID NO:4 (or 5) and a VL domain comprising the sequence of SEQ IDNO:6 (or 7).

In certain embodiments, each anti-IGF-1R binding site of a PBA comprisesa VH domain comprising the aa sequence of SEQ ID NO:1, wherein X1 is notT, X2 is not V, X6 is not R, X8 is not D or X10 is not I, or a VL domaincomprising the aa sequence of SEQ ID NO:3 or each anti-ErbB3 bindingsite of a PBA comprises a VH domain comprising the aa sequence of SEQ IDNO:5 or a VL domain comprising the sequence of SEQ ID NO:7. In certainembodiments, each anti-IGF-1R binding site of a PBA comprises a VHdomain comprising the aa sequence of SEQ ID NO:1, wherein X1 is not T,X2 is not V, X6 is not R, X8 is not D or X10 is not I, a VL domaincomprising the sequence of SEQ ID NO:3; and each anti-ErbB3 binding siteof the PBA comprises a VH domain comprising the aa sequence of SEQ IDNO:5 and a VL domain comprising the sequence of SEQ ID NO:7.

In certain embodiments, each anti-IGF-1R binding site of a PBA comprisesa VH domain comprising an aa sequence selected from the group consistingof SEQ ID NOs: 8-31 and/or a VL domain comprising an aa sequenceselected from the group consisting of SEQ ID NOs: 32-133 and/or eachanti-ErbB3 binding site comprises a VH aa sequence selected from thegroup consisting of SEQ ID NOs: 134-165; and/or a VL aa sequenceselected from the group consisting of SEQ ID NOs: 166-200. In certainembodiments, (a) each first VH domain comprises an aa sequence selectedfrom the group comprising SEQ ID NOs: 8-31, each first VL domaincomprises an aa sequence selected from the group consisting of SEQ IDNOs: 32-133, each second VH domain comprises an aa sequence selectedfrom the group consisting of SEQ ID NOs: 134-165 and each second VLdomain comprises an aa sequence selected from the group comprising SEQID NOs: 166-200, or (b) each first VH domain comprises an aa sequenceselected from the group comprising SEQ ID NOs: 134-165, each first VLdomain comprises an aa sequence selected from the group consisting ofSEQ ID NOs: 166-200, each second VH domain comprises an aa sequenceselected from the group comprising SEQ ID NOs: 8-31 and each second VLdomain consisting of an aa sequence selected from the group comprisingSEQ ID NOs: 32-133.

In certain embodiments, each anti-IGF-1R VH domain of a PBA comprisesthe aa sequence of SEQ ID NO:8 and each anti-IGF-1R VL domain of the PBAcomprises the aa sequence of SEQ ID NO:32. In certain embodiments, eachanti-IGF-1R VH domain of a PBA comprises the aa sequence of SEQ ID NO:9and each anti-IGF-1R VL domain of the PBA comprises the aa sequence ofSEQ ID NO:33. In certain embodiments, each anti-IGF-1R VH domain of aPBA comprises the aa sequence of SEQ ID NO:10 and each anti-IGF-1R VLdomain of the PBA comprises the aa sequence of SEQ ID NO:34. In certainembodiments, each anti-IGF-1R VH domain of a PBA comprises the aasequence of SEQ ID NO:11 and each anti-IGF-1R VL domain of the PBAcomprises the aa sequence of SEQ ID NO:35. In certain embodiments, eachanti-IGF-1R VH domain of a PBA comprises the aa sequence of SEQ ID NO:8and each anti-IGF-1R VL domain of the PBA comprises the aa sequence ofSEQ ID NO:33. In certain embodiments, each anti-IGF-1R VH domain of aPBA comprises the aa sequence of SEQ ID NO:10 and each anti-IGF-1R VLdomain of the PBA comprises the aa sequence of SEQ ID NO:32.

In certain embodiments, each anti-ErbB3 VH domain of a PBA comprises theaa sequence of SEQ ID NO:134 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:166. In certain embodiments, eachanti-ErbB3 VH domain of a PBA comprises the aa sequence of SEQ ID NO:135and the anti-ErbB3 VL domain of the PBA comprise the aa sequence of SEQID NO:167. In certain embodiments, each anti-ErbB3 VH domain of a PBAcomprises the aa sequence of SEQ ID NO:136 and each anti-ErbB3 VL domainof the PBA comprises the aa sequence of SEQ ID NO:168. In certainembodiments, each anti-ErbB3 VH domain of a PBA comprises the aasequence of SEQ ID NO:137 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:169. In certain embodiments, eachanti-ErbB3 VH domain of a PBA comprises the aa sequence of SEQ ID NO:138and each anti-ErbB3 VL domain of the PBA comprises the aa sequence ofSEQ ID NO:170. In certain embodiments, each anti-ErbB3 VH domain of aPBA comprises the aa sequence of SEQ ID NO:139 and each anti-ErbB3 VLdomain of the PBA comprises the aa sequence of SEQ ID NO:171. In certainembodiments, each anti-ErbB3 VH domain of a PBA comprises the aasequence of SEQ ID NO:140 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:172. In certain embodiments, eachanti-ErbB3 VH domain of a PBA comprises the aa sequence of SEQ ID NO:141and each anti-ErbB3 VL domain of the PBA comprises the aa sequence ofSEQ ID NO:173. In certain embodiments, each anti-ErbB3 VH domain of aPBA comprises the aa sequence of SEQ ID NO:142 and each anti-ErbB3 VLdomain of the PBA comprises the aa sequence of SEQ ID NO:174. In certainembodiments, each anti-ErbB3 VH domain of a PBA comprises the aasequence of SEQ ID NO:143 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:175. In certain embodiments, eachanti-ErbB3 VH domain of a PBA comprises the aa sequence of SEQ ID NO:136and each anti-ErbB3 VL domain of the PBA comprises the aa sequence ofSEQ ID NOs: 169.

In certain embodiments, each anti-IGF-1R VH domain of a PBA comprisesthe aa sequence of SEQ ID NO:8 and each anti-IGF-1R VL domain of the PBAcomprises the aa sequence of SEQ ID NO:32; and (a) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:134 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:166; or (b) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:135 and the anti-ErbB3 VL domain of the PBAcomprise the aa sequence of SEQ ID NO:167; or (c) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:136 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:168; or (d) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:137 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:169; or (e) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:138 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:170; or (f) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:139 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:171; or (g) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:140 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:172; or (h) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:141 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:173; or (i) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:142 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:174; or (j) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:143 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:175; or (k) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:136 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ ID NOs:169.

In certain embodiments, each anti-IGF-1R VH domain of a PBA comprisesthe aa sequence of SEQ ID NO:9 and each anti-IGF-1R VL domain of the PBAcomprises the aa sequence of SEQ ID NO:33; and (a) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:134 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:166; or (b) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:135 and the anti-ErbB3 VL domain of the PBAcomprise the aa sequence of SEQ ID NO:167; or (c) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:136 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:168; or (d) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:137 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:169; or (e) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:138 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:170; or (f) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:139 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:171; or (g) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:140 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:172; or (h) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:141 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:173; or (i) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:142 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:174; or (j) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:143 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:175; or (k) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:136 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ ID NOs:169.

In certain embodiments, each anti-IGF-1R VH domain comprises the aasequence of SEQ ID NO:10 and each anti-IGF-1R VL domain comprises the aasequence of SEQ ID NO:34; and (a) each anti-ErbB3 VH domain of the PBAcomprises the aa sequence of SEQ ID NO:134 and each anti-ErbB3 VL domainof the PBA comprises the aa sequence of SEQ ID NO:166; or (b) eachanti-ErbB3 VH domain of the PBA comprises the aa sequence of SEQ IDNO:135 and the anti-ErbB3 VL domain of the PBA comprise the aa sequenceof SEQ ID NO:167; or (c) each anti-ErbB3 VH domain of the PBA comprisesthe aa sequence of SEQ ID NO:136 and each anti-ErbB3 VL domain of thePBA comprises the aa sequence of SEQ ID NO:168; or (d) each anti-ErbB3VH domain of the PBA comprises the aa sequence of SEQ ID NO:137 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:169; or (e) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:138 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:170; or (f) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:139 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:171; or (g) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:140 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:172; or (h) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:141 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:173; or (i) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:142 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:174; or (j) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:143 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:175; or (k) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:136 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NOs: 169.

In certain embodiments, each anti-IGF-1R VH domain of a PBA comprisesthe aa sequence of SEQ ID NO:11 and each anti-IGF-1R VL domain of thePBA comprises the aa sequence of SEQ ID NO:35; and (a) each anti-ErbB3VH domain of the PBA comprises the aa sequence of SEQ ID NO:134 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:166; or (b) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:135 and the anti-ErbB3 VL domain of the PBAcomprise the aa sequence of SEQ ID NO:167; or (c) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:136 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:168; or (d) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:137 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:169; or (e) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:138 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:170; or (f) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:139 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:171; or (g) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:140 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:172; or (h) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:141 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:173; or (i) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:142 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:174; or (j) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:136 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NOs: 169.

In certain embodiments, each anti-IGF-1R VH domain of the PBA comprisesthe aa sequence of SEQ ID NO:8 and each anti-IGF-1R VL domain of the PBAcomprises the aa sequence of SEQ ID NO:33; and (a) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:134 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:166; or (b) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:135 and the anti-ErbB3 VL domain of the PBAcomprise the aa sequence of SEQ ID NO:167; or (c) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:136 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:168; or (d) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:137 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:169; or (e) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:138 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:170; or (f) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:139 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:171; or (g) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:140 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:172; or (h) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:141 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:173; or (i) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:142 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:174; or (j) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:143 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:175; or (k) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:136 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ ID NOs:169.

In certain embodiments, each anti-IGF-1R VH domain of a PBA comprisesthe aa sequence of SEQ ID NO:10 and each anti-IGF-1R VL domain of thePBA comprises the aa sequence of SEQ ID NO:32; and (a) each anti-ErbB3VH domain of the PBA comprises the aa sequence of SEQ ID NO:134 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:166; or (b) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:135 and the anti-ErbB3 VL domain of the PBAcomprise the aa sequence of SEQ ID NO:167; or (c) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:136 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:168; or (d) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:137 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:169; or (e) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:138 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:170; or (f) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:139 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:171; or (g) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:140 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:172; or (h) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:141 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:173; or (i) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:142 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ IDNO:174; or (j) each anti-ErbB3 VH domain of the PBA comprises the aasequence of SEQ ID NO:143 and each anti-ErbB3 VL domain of the PBAcomprises the aa sequence of SEQ ID NO:175; or (k) each anti-ErbB3 VHdomain of the PBA comprises the aa sequence of SEQ ID NO:136 and eachanti-ErbB3 VL domain of the PBA comprises the aa sequence of SEQ ID NOs:169.

In certain embodiments, (a) each heavy chain of a PBA comprises an aasequence selected from the group consisting of SF-G1-P1 (SEQ ID NO:212);SF-G1-M1.3 (SEQ ID NO:214); SF-G1-M27 (SEQ ID NO:216); SF-G1-P6 (SEQ IDNO:218); SF-G1-B69 (SEQ ID NO:220); P4-G1-C8 (SEQ ID NO:222); P4-G1-P1(SEQ ID NO:224); P4-G1-M1.3 (SEQ ID NO:226); P4-G1-M27 (SEQ ID NO:228);P4-G1-P6 (SEQ ID NO:230); P4-G1-B69 (SEQ ID NO:232); M78-G1-C8 (SEQ IDNO:234); M78-G1-P1 (SEQ ID NO:236); M78-G1-M1.3 (SEQ ID NO:238);M78-G1-M27 (SEQ ID NO:240); M78-G1-P6 (SEQ ID NO:242); M78-G1-B69 (SEQID NO:244); M57-G1-C8 (SEQ ID NO:246); M57-G1-P1 (SEQ ID NO:248);M57-G1-M1.3 (SEQ ID NO:250); M57-G1-M27 (SEQ ID NO:252); M57-G1-P6 (SEQID NO:254) and M57-G1-B69 (SEQ ID NO:256) and/or each light chain of thePBA comprises an aa sequence selected from the group consisting of SFkappa light chain (SEQ ID NO:202); P4 kappa light chain (SEQ ID NO:204);M78 kappa light chain (SEQ ID NO:206); and M57 kappa light chain (SEQ IDNO:208); or (b) each heavy chain of a PBA comprises an aa sequenceselected from the group comprising P1-G1-P4 (SEQ ID NO:268); P1-G1-M57(SEQ ID NO:270); P1-G1-M78 (SEQ ID NO:272); M27-G1-P4 (SEQ ID NO:274);M27-G1-M57 (SEQ ID NO:276); M27-G1-M78 (SEQ ID NO:278); M7-G1-P4 (SEQ IDNO:280); M7-G1-M57 ((SEQ ID NO:282); M7-G1-M78 (SEQ ID NO:284);B72-G1-P4 (SEQ ID NO:286); B72-G1-M57 (SEQ ID NO:288); B72-G1-M78 (SEQID NO:290); B60-G1-P4 (SEQ ID NO:292); B60-G1-M57 (SEQ ID NO:294);B60-G1-M78 (SEQ ID NO:296); B60-G2-M78 (SEQ ID NO:355) and M7-G2-M78(SEQ ID NO:357) and/or each light chain of the PBA comprises an aasequence selected from the group consisting of P1 lambda light chain(SEQ ID NO:258); M27 lambda light chain (SEQ ID NO:260); M7 lambda lightchain (SEQ ID NO:262); B72 lambda light chain (SEQ ID NO:264); and B60lambda light chain (SEQ ID NO:266).

In certain embodiments, (a) each heavy chain of a PBA comprises an aasequence differing in at least one aa addition, deletion or substitutionfrom an aa sequence selected from the group consisting of SF-G1-P1 (SEQID NO:212); SF-G1-M1.3 (SEQ ID NO:214); SF-G1-M27 (SEQ ID NO:216);SF-G1-P6 (SEQ ID NO:218); SF-G1-B69 (SEQ ID NO:220); P4-G1-C8 (SEQ IDNO:222); P4-G1-P1 (SEQ ID NO:224); P4-G1-M1.3 (SEQ ID NO:226); P4-G1-M27(SEQ ID NO:228); P4-G1-P6 (SEQ ID NO:230); P4-G1-B69 (SEQ ID NO:232);M78-G1-C8 (SEQ ID NO:234); M78-G1-P1 (SEQ ID NO:236); M78-G1-M1.3 (SEQID NO:238); M78-G1-M27 (SEQ ID NO:240); M78-G1-P6 (SEQ ID NO:242);M78-G1-B69 (SEQ ID NO:244); M57-G1-C8 (SEQ ID NO:246); M57-G1-P1 (SEQ IDNO:248); M57-G1-M1.3 (SEQ ID NO:250); M57-G1-M27 (SEQ ID NO:252);M57-G1-P6 (SEQ ID NO:254) and M57-G1-B69 (SEQ ID NO:256) and each lightchain of the PBA comprises an aa sequence selected from the groupconsisting of SF kappa light chain (SEQ ID NO:202); P4 kappa light chain(SEQ ID NO:204); M78 kappa light chain (SEQ ID NO:206); and M57 kappalight chain (SEQ ID NO:208); or (b) each heavy chain of a PBA comprisesan aa sequence selected from the group consisting of SF-G1-P1 (SEQ IDNO:212); SF-G1-M1.3 (SEQ ID NO:214); SF-G1-M27 (SEQ ID NO:216); SF-G1-P6(SEQ ID NO:218); SF-G1-B69 (SEQ ID NO:220); P4-G1-C8 (SEQ ID NO:222);P4-G1-P1 (SEQ ID NO:224); P4-G1-M1.3 (SEQ ID NO:226); P4-G1-M27 (SEQ IDNO:228); P4-G1-P6 (SEQ ID NO:230); P4-G1-B69 (SEQ ID NO:232); M78-G1-C8(SEQ ID NO:234); M78-G1-P1 (SEQ ID NO:236); M78-G1-M1.3 (SEQ ID NO:238);M78-G1-M27 (SEQ ID NO:240); M78-G1-P6 (SEQ ID NO:242); M78-G1-B69 (SEQID NO:244); M57-G1-C8 (SEQ ID NO:246); M57-G1-P1 (SEQ ID NO:248);M57-G1-M1.3 (SEQ ID NO:250); M57-G1-M27 (SEQ ID NO:252); M57-G1-P6 (SEQID NO:254) and M57-G1-B69 (SEQ ID NO:256); and each light chain of thePBA comprises an aa sequence differing in at least one aa addition,deletion or substitution from an aa sequence selected from the groupconsisting of SF kappa light chain (SEQ ID NO:202); P4 kappa light chain(SEQ ID NO:204); M78 kappa light chain (SEQ ID NO:206); and M57 kappalight chain (SEQ ID NO:208); or (c) each heavy chain of a PBA comprisesan aa sequence differing in at least one aa addition, deletion orsubstitution from an aa sequence selected from the group comprisingP1-G1-P4 (SEQ ID NO:268); P1-G1-M57 (SEQ ID NO:270); P1-G1-M78 (SEQ IDNO:272); M27-G1-P4 (SEQ ID NO:274); M27-G1-M57 (SEQ ID NO:276);M27-G1-M78 (SEQ ID NO:278); M7-G1-P4 (SEQ ID NO:280); M7-G1-M57 ((SEQ IDNO:282); M7-G1-M78 (SEQ ID NO:284); B72-G1-P4 (SEQ ID NO:286);B72-G1-M57 (SEQ ID NO:288); B72-G1-M78 (SEQ ID NO:290); B60-G1-P4 (SEQID NO:292); B60-G1-M57 (SEQ ID NO:294); B60-G1-M78 (SEQ ID NO:296);B60-G2-M78 (SEQ ID NO:355) and M7-G2-M78 (SEQ ID NO:357) and each lightchain of the PBA comprises an aa sequence selected from the groupconsisting of P1 lambda light chain (SEQ ID NO:258); M27 lambda lightchain (SEQ ID NO:260); M7 lambda light chain (SEQ ID NO:262); B72 lambdalight chain (SEQ ID NO:264); and B60 lambda light chain (SEQ ID NO:266);or (d) each heavy chain of a PBA comprises an aa sequence selected fromthe group comprising P1-G1-P4 (SEQ ID NO:268); P1-G1-M57 (SEQ IDNO:270); P1-G1-M78 (SEQ ID NO:272); M27-G1-P4 (SEQ ID NO:274);M27-G1-M57 (SEQ ID NO:276); M27-G1-M78 (SEQ ID NO:278); M7-G1-P4 (SEQ IDNO:280); M7-G1-M57 ((SEQ ID NO:282); M7-G1-M78 (SEQ ID NO:284);B72-G1-P4 (SEQ ID NO:286); B72-G1-M57 (SEQ ID NO:288); B72-G1-M78 (SEQID NO:290); B60-G1-P4 (SEQ ID NO:292); B60-G1-M57 (SEQ ID NO:294);B60-G1-M78 (SEQ ID NO:296); B60-G2-M78 (SEQ ID NO:355) and M7-G2-M78(SEQ ID NO:357) and each light chain of the PBA comprises an aa sequencediffering in at least one aa addition, deletion or substitution from anaa sequence selected from the group consisting of P1 lambda light chain(SEQ ID NO:258); M27 lambda light chain (SEQ ID NO:260); M7 lambda lightchain (SEQ ID NO:262); B72 lambda light chain (SEQ ID NO:264); and B60lambda light chain (SEQ ID NO:266), wherein the PBA differs from 16F inat least one aa, CDR or variable domain.

In certain embodiments, (a) each heavy chain of a PBA, bound to theother heavy chain of the PBA by at least one bond, comprises an aasequence that is at least 90% identical to one of the following aasequences or differs from one of the following aa sequences in 1-30 aasubstitutions, deletions and/or additions: SF-G1-P1 (SEQ ID NO:212);SF-G1-M1.3 (SEQ ID NO:214); SF-G1-M27 (SEQ ID NO:216); SF-G1-P6 (SEQ IDNO:218); SF-G1-B69 (SEQ ID NO:220); P4-G1-C8 (SEQ ID NO:222); P4-G1-P1(SEQ ID NO:224); P4-G1-M1.3 (SEQ ID NO:226); P4-G1-M27 (SEQ ID NO:228);P4-G1-P6 (SEQ ID NO:230); P4-G1-B69 (SEQ ID NO:232); M78-G1-C8 (SEQ IDNO:234); M78-G1-P1 (SEQ ID NO:236); M78-G1-M1.3 (SEQ ID NO:238);M78-G1-M27 (SEQ ID NO:240); M78-G1-P6 (SEQ ID NO:242); M78-G1-B69 (SEQID NO:244); M57-G1-C8 (SEQ ID NO:246); M57-G1-P1 (SEQ ID NO:248);M57-G1-M1.3 (SEQ ID NO:250); M57-G1-M27 (SEQ ID NO:252); M57-G1-P6 (SEQID NO:254) and M57-G1-B69 (SEQ ID NO:256) and (b) each light chain ofthe PBA, bound to one heavy chain of (a) by at least one bond, comprisesan aa sequence that is at least 90% identical to one of the following aasequences or differs from one of the following aa sequences in 1-30 aasubstitutions, deletions and/or additions: SF kappa light chain (SEQ IDNO:202); P4 kappa light chain (SEQ ID NO:204); M78 kappa light chain(SEQ ID NO:206); and M57 kappa light chain (SEQ ID NO:208); or (c) eachheavy chain of a PBA, bound to the other heavy chain of the PBA by atleast one bond, comprises an aa sequence that is at least 90% identicalto one of the following aa sequences or differs from one of thefollowing aa sequences in 1-30 aa substitutions, deletions and/oradditions: P1-G1-P4 (SEQ ID NO:268); P1-G1-M57 (SEQ ID NO:270);P1-G1-M78 (SEQ ID NO:272); M27-G1-P4 (SEQ ID NO:274); M27-G1-M57 (SEQ IDNO:276); M27-G1-M78 (SEQ ID NO:278); M7-G1-P4 (SEQ ID NO:280); M7-G1-M57((SEQ ID NO:282); M7-G1-M78 (SEQ ID NO:284); B72-G1-P4 (SEQ ID NO:286);B72-G1-M57 (SEQ ID NO:288); B72-G1-M78 (SEQ ID NO:290); B60-G1-P4 (SEQID NO:292); B60-G1-M57 (SEQ ID NO:294); and B60-G1-M78 (SEQ IDNO:296),); B60-G2-M78 (SEQ ID NO:355) and M7-G2-M78 (SEQ ID NO:357) and(d) each light chain of the PBA, bound to one heavy chain of (c) by atleast one bond, comprises an aa sequence that is at least 90% identicalto one of the following aa sequences or which differs from one of thefollowing aa sequences in 1-30 aa substitutions, deletions and/oradditions: P1 lambda light chain (SEQ ID NO:258); M27 lambda light chain(SEQ ID NO:260); M7 lambda light chain (SEQ ID NO:262); B72 lambda lightchain (SEQ ID NO:264); and B60 lambda light chain (SEQ ID NO:266).

In certain embodiments, (a) each heavy chain of a PBA comprises an aasequence that is at least 95% identical to one of the following aasequence or differs from one of the following aa sequences in 1-10 aasubstitutions, deletions and/or additions: SF-G1-P1 (SEQ ID NO:212);SF-G1-M1.3 (SEQ ID NO:214); SF-G1-M27 (SEQ ID NO:216); SF-G1-P6 (SEQ IDNO:218); SF-G1-B69 (SEQ ID NO:220); P4-G1-C8 (SEQ ID NO:222); P4-G1-P1(SEQ ID NO:224); P4-G1-M1.3 (SEQ ID NO:226); P4-G1-M27 (SEQ ID NO:228);P4-G1-P6 (SEQ ID NO:230); P4-G1-B69 (SEQ ID NO:232); M78-G1-C8 (SEQ IDNO:234); M78-G1-P1 (SEQ ID NO:236); M78-G1-M1.3 (SEQ ID NO:238);M78-G1-M27 (SEQ ID NO:240); M78-G1-P6 (SEQ ID NO:242); M78-G1-B69 (SEQID NO:244); M57-G1-C8 (SEQ ID NO:246); M57-G1-P1 (SEQ ID NO:248);M57-G1-M1.3 (SEQ ID NO:250); M57-G1-M27 (SEQ ID NO:252); M57-G1-P6 (SEQID NO:254) and M57-G1-B69 (SEQ ID NO:256), and (b) each light chain ofthe PBA comprises an aa sequence that is at least 95% identical to oneof the following aa sequences or differs from one of the following aasequences in 1-10 aa substitutions, deletions and/or additions: SF kappalight chain (SEQ ID NO:202); P4 kappa light chain (SEQ ID NO:204); M78kappa light chain (SEQ ID NO:206); and M57 kappa light chain (SEQ IDNO:208); or (c) each heavy chain of a PBA comprises an aa sequence thatis at least 95% identical to one of the following aa sequences ordiffers from one of the following aa sequences in 1-10 aa substitutions,deletions and/or additions: P1-G1-P4 (SEQ ID NO:268); P1-G1-M57 (SEQ IDNO:270); P1-G1-M78 (SEQ ID NO:272); M27-G1-P4 (SEQ ID NO:274);M27-G1-M57 (SEQ ID NO:276); M27-G1-M78 (SEQ ID NO:278); M7-G1-P4 (SEQ IDNO:280); M7-G1-M57 ((SEQ ID NO:282); M7-G1-M78 (SEQ ID NO:284);B72-G1-P4 (SEQ ID NO:286); B72-G1-M57 (SEQ ID NO:288); B72-G1-M78 (SEQID NO:290); B60-G1-P4 (SEQ ID NO:292); B60-G1-M57 (SEQ ID NO:294); andB60-G1-M78 (SEQ ID NO:296); B60-G2-M78 (SEQ ID NO:355) and M7-G2-M78(SEQ ID NO:357) and (d) each light chain of the PBA comprises an aasequence that is at least 95% identical to one of the following aasequences or differs from one of the following aa sequences in 1-10 aasubstitutions, deletions and/or additions: P1 lambda light chain (SEQ IDNO:258); M27 lambda light chain (SEQ ID NO:260); M7 lambda light chain(SEQ ID NO:262); B72 lambda light chain (SEQ ID NO:264); B60 lambdalight chain (SEQ ID NO:266).

Exemplary PBA include the following: (a) an SF-G1-P1 PBA comprising: twoheavy chains, each comprising a heavy chain aa sequence of SEQ IDNO:212; and two light chains, each comprising a light chain sequence ofSEQ ID NO:202; (b) An SF-G1-M1.3 PBA comprising: two heavy chains, eachcomprising a heavy chain aa sequence of SEQ ID NO:214; and two lightchains, each comprising a light chain aa sequence of SEQ ID NO:202; (c)an SF-G1-M27 PBA comprising: two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:216; and two light chains, eachcomprising a light chain aa sequence of SEQ ID NO:202; (d) an SF-G1-P6PBA comprising: two heavy chains, each comprising a heavy chain aasequence of SEQ ID NO:218; and two light chains, each comprising a lightchain aa sequence of SEQ ID NO:202; (e) an SF-G1-B69 PBA comprising: twoheavy chains, each comprising a heavy chain aa sequence of SEQ IDNO:220; and two light chains, each comprising a light chain aa sequenceof SEQ ID NO:202; (f) a P4-G1-C8 PBA comprising: two heavy chains, eachcomprising a heavy chain aa sequence of SEQ ID NO:222; and two lightchains, each comprising a light chain aa sequence of SEQ ID NO:204 (g) aP4-G1-P1 PBA comprising: two heavy chains, each comprising a heavy chainaa sequence of SEQ ID NO:224; and two light chains, each comprising alight chain aa sequence of SEQ ID NO:204; (h) a P4-G1-M1.3 PBAcomprising: two heavy chains, each comprising a heavy chain aa sequenceof SEQ ID NO:226; and two light chains, each comprising a light chain aasequence of SEQ ID NO:204; (i) a P4-G1-M27 PBA comprising: two heavychains, each comprising a heavy chain aa sequence of SEQ ID NO:228; andtwo light chains, each comprising a light chain aa sequence of SEQ IDNO:204; (j) a P4-G1-P6 PBA comprising: two heavy chains, each comprisinga heavy chain aa sequence of SEQ ID NO:230; and two light chains, eachcomprising a light chain aa sequence of SEQ ID NO:204; (h) a P4-G1-B69PBA comprising: two heavy chains, each comprising a heavy chain aasequence of SEQ ID NO:232; and two light chains, each comprising a lightchain aa sequence of SEQ ID NO:204; (i) an M78-G1-C8 PBA comprising: twoheavy chains, each comprising a heavy chain aa sequence of SEQ IDNO:234; and two light chains, each comprising a light chain aa sequenceof SEQ ID NO:206; (j) an M78-G1-P1 PBA comprising: two heavy chains,each comprising a heavy chain aa sequence of SEQ ID NO:236; and twolight chains, each comprising a light chain aa sequence of SEQ IDNO:206; (k) an M78-G1-M1.3 PBA comprising: two heavy chains, eachcomprising a heavy chain aa sequence of SEQ ID NO:238; and two lightchains, each comprising a light chain aa sequence of SEQ ID NO:206; (l)an M78-G1-M27 PBA comprising: two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:240; and two light chains, eachcomprising a light chain aa sequence of SEQ ID NO:206; (m) an M78-G1-P6PBA comprising: two heavy chains, each comprising a heavy chain aasequence of SEQ ID NO:242; and two light chains, each comprising a lightchain aa sequence of SEQ ID NO:206; (n) an M78-G1-B69 PBA comprising:two heavy chains, each comprising a heavy chain aa sequence of SEQ IDNO:244; and two light chains, each comprising a light chain aa sequenceof SEQ ID NO:206; (o) an M57-G1-C8 PBA comprising: two heavy chains,each comprising a heavy chain aa sequence of SEQ ID NO:246; and twolight chains, each comprising a light chain aa sequence of SEQ IDNO:208; (p) an M57-G1-P1 PBA comprising: two heavy chains, eachcomprising a heavy chain aa sequence of SEQ ID NO:248; and two lightchains, each comprising a light chain aa sequence of SEQ ID NO:208; (r)an M57-G1-M1.3 PBA comprising: two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:250; and two light chains, eachcomprising a light chain aa sequence of SEQ ID NO:208; (s) an M57-G1-M27PBA comprising: two heavy chains, each comprising a heavy chain aasequence of SEQ ID NO:252; and two light chains, each comprising a lightchain aa sequence of SEQ ID NO:208; (t) an M57-G1-P6 PBA comprising: twoheavy chains, each comprising a heavy chain aa sequence of SEQ IDNO:254; and two light chains, each comprising a light chain aa sequenceof SEQ ID NO:208; (u) an M57-G1-B69 PBA comprising: two heavy chains,each comprising a heavy chain aa sequence of SEQ ID NO:256; and twolight chains, each comprising a light chain aa sequence of SEQ IDNO:208; (v) a P1-G1-P4 PBA comprising: two heavy chains, each comprisinga heavy chain aa sequence of SEQ ID NO:268; and two light chains, eachcomprising a light chain aa sequence of SEQ ID NO:258; (w) a P1-G1-M57PBA comprising: two heavy chains, each comprising a heavy chain aasequence of SEQ ID NO:270; and two light chains, each comprising a lightchain aa sequence of SEQ ID NO:258; (x) a P1-G1-M78 PBA comprising: twoheavy chains, each comprising a heavy chain aa sequence of SEQ IDNO:272; and two light chains, each comprising a light chain aa sequenceof SEQ ID NO:258; (y) an M27-G1-P4 PBA comprising: two heavy chains,each comprising a heavy chain aa sequence of SEQ ID NO:274; and twolight chains, each comprising a light chain aa sequence of SEQ IDNO:260; (z) an M27-G1-M57 PBA comprising: two heavy chains, eachcomprising a heavy chain aa sequence of SEQ ID NO:276; and two lightchains, each comprising a light chain aa sequence of SEQ ID NO:260; (aa)an M27-G1-M78 PBA comprising: two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:278; and two light chains, eachcomprising a light chain aa sequence of SEQ ID NO:260; (ab) an M7-G1-P4PBA comprising: two heavy chains, each comprising a heavy chain aasequence of SEQ ID NO:280; and two light chains, each comprising a lightchain aa sequence of SEQ ID NO:262; (ac) an M7-G1-M57 PBA comprising:two heavy chains, each comprising a heavy chain aa sequence of SEQ IDNO:282; and two light chains, each comprising a light chain aa sequenceof SEQ ID NO:262; (ad) an M7-G1-M78 PBA comprising: two heavy chains,each comprising a heavy chain aa sequence of SEQ ID NO:284; and twolight chains, each comprising a light chain aa sequence of SEQ IDNO:262; (ae) a B72-G1-P4 PBA comprising: two heavy chains, eachcomprising a heavy chain aa sequence of SEQ ID NO:286; and two lightchains, each comprising a light chain aa sequence of SEQ ID NO:264; (af)a B72-G1-M57 PBA comprising: two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:288; and two light chains, eachcomprising a light chain aa sequence of SEQ ID NO:264; (ag) a B72-G1-M78PBA comprising: two heavy chains, each comprising a heavy chain aasequence of SEQ ID NO:290; and two light chains, each comprising a lightchain aa sequence of SEQ ID NO:264; (ah) a B60-G1-P4 PBA comprising: twoheavy chains, each comprising a heavy chain aa sequence of SEQ IDNO:292; and two light chains, each comprising a light chain aa sequenceof SEQ ID NO:266; (ai) a B60-G1-M57 PBA comprising: two heavy chains,each comprising a heavy chain aa sequence of SEQ ID NO:294; and twolight chains, each comprising a light chain aa sequence of SEQ IDNO:266; and (aj) a B60-G1-M78 PBA comprising: two heavy chains, eachcomprising a heavy chain aa sequence of SEQ ID NO:296; and two lightchains, each comprising a light chain aa sequence of SEQ ID NO:266.

Also provided herein are monospecific antibodies. In certainembodiments, an anti-IGF-1R monoclonal antibody comprises a firstsequence comprising in amino to carboxy order a VLCDR1 sequence, aVLCDR2 sequence and a VLCDR3 sequence of SF kappa light chain asindicated by dotted underlining in FIG. 5A, SEQ ID NO:202, antibodyfurther comprising a second sequence comprising in amino to carboxyorder a VHCDR1 sequence, a VHCDR2 sequence and a VHCDR3 sequence of SFheavy chain as indicated by the first three dotted underlined sequencesrespectively in FIG. 5A, SEQ ID NO:210, wherein the first sequence andthe second sequence are non-overlapping. In certain embodiments, ananti-IGF-1R monoclonal antibody comprises in amino to carboxy order aVLCDR1 sequence, a VLCDR2 sequence and a VLCDR3 sequence of P4 kappalight chain as indicated by dotted underlining in FIG. 5A, SEQ IDNO:204, antibody further comprising in amino to carboxy order a VHCDR1sequence, a VHCDR2 sequence and a VHCDR3 sequence of P4 heavy chain asindicated by the first three dotted underlined sequences respectively inFIG. 5A, SEQ ID NO:222. In certain embodiments, an anti-IGF-1Rmonoclonal antibody comprises in amino to carboxy order a VLCDR1sequence, a VLCDR2 sequence and a VLCDR3 sequence of M78 kappa lightchain as indicated by dotted underlining in FIG. 5A, SEQ ID NO:206,antibody further comprising in amino to carboxy order a VHCDR1 sequence,a VHCDR2 sequence and a VHCDR3 sequence of M78 heavy chain as indicatedby the first three dotted underlined sequences respectively in FIG. 5A,SEQ ID NO:234. In certain embodiments, an anti-IGF-1R monoclonalantibody comprising in amino to carboxy order a VLCDR1 sequence, aVLCDR2 sequence and a VLCDR3 sequence of M57 kappa light chain asindicated by dotted underlining in FIG. 5A, SEQ ID NO:208, antibodyfurther comprising in amino to carboxy order a VHCDR1 sequence, a VHCDR2sequence and a VHCDR3 sequence of M57 heavy chain as indicated by thefirst three dotted underlined sequences respectively in FIG. 5A, SEQ IDNO:246.

In certain embodiments, an anti-IGF-1R antibody comprises a VH domaincomprising a set of three VH CDRs comprising VHCDR1, VHCDR2, VHCDR3,and/or a VL domain comprising a set of three VL CDRs comprising VLCDR1,VLCDR2 and VLCDR3, CDRs comprising the sequences of SEQ ID NOs: 302,303, 304, 305, 306, and 307, respectively, and each CDR furthercomprising an amino terminus and a carboxy terminus, wherein the CDRs ofeach set of CDRs are arranged in the antibody in a linear amino tocarboxy order of CDR1, CDR2, and CDR3, wherein the CDRs comprisevariable aas, which independently represent any aa set forth at thecorresponding position in FIG. 1 (VH) or FIG. 2 (VL), and the antibodydoes not comprise the SF module. In certain embodiments, the VHCDR1,VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 of an anti-IGF-1R antibodycomprise the sequences of SEQ ID NOs: 302, 303, 304, 305, 306, and 308,respectively. In certain embodiments, the VHCDR1, VHCDR2 and VHCDR3domains of an anti-IGF-1R antibody comprise the corresponding aasequences of any one of SEQ ID NOs: 8-10 and 12-31, and the VLCDR1,VLCDR2 and VLCDR3 domains of the anti-IGF-1R antibody comprise thecorresponding aa sequences of any one of SEQ ID NOs: 32-34 and 36-133.In certain embodiments, (a) the VHCDR1, VHCDR2, VHCDR3 of an anti-IGF-1Rantibody comprise the aa sequence of the corresponding CDRs of SEQ IDNO:8 and the VLCDR1, VLCDR2 and VLCDR3 of the anti-IGF-1R antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:32; or(b) the VHCDR1, VHCDR2, VHCDR3 of the anti-IGF-1R antibody comprise theaa sequence of the corresponding CDRs of SEQ ID NO:9 and the VLCDR1,VLCDR2 and VLCDR3 of the anti-IGF-1R antibody comprise the aa sequenceof the corresponding CDRs of SEQ ID NO:33; or (c) the VHCDR1, VHCDR2,VHCDR3 of the anti-IGF-1R antibody comprise the aa sequence of thecorresponding CDRs of SEQ ID NO:10 and the VLCDR1, VLCDR2 and VLCDR3 ofthe anti-IGF-1R antibody comprise the aa sequence of the correspondingCDRs of SEQ ID NO:34; or (d) the VHCDR1, VHCDR2, VHCDR3 of theanti-IGF-1R antibody comprise the aa sequence of the corresponding CDRsof SEQ ID NO:11 and the VLCDR1, VLCDR2 and VLCDR3 of the anti-IGF-1Rantibody comprise the aa sequence of the corresponding CDRs of SEQ IDNO:35; or (e) the VHCDR1, VHCDR2, VHCDR3 of the anti-IGF-1R antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:8 andthe VLCDR1, VLCDR2 and VLCDR3 of the anti-IGF-1R antibody comprise theaa sequence of the corresponding CDRs of SEQ ID NO:33; or (f) theVHCDR1, VHCDR2, VHCDR3 of the anti-IGF-1R antibody comprise the aasequence of the corresponding CDRs of SEQ ID NO:10 and the VLCDR1,VLCDR2 and VLCDR3 of the anti-IGF-1R antibody comprise the aa sequenceof the corresponding CDRs of SEQ ID NO:32.

In certain embodiments, the VH domain of an anti-IGF-1R antibodycomprises the aa sequence of SEQ ID NO:1, which sequence comprisesvariable aas, which independently represent any aa set forth at thecorresponding position in FIG. 1; the VL domain of the anti-IGF-1Rantibody comprises the aa sequence of SEQ ID NO:2 (or 3), which sequencecomprises variable aas, which independently represent any aa set forthat the corresponding position in FIG. 2; or the VH domain of theanti-IGF-1R antibody comprises the aa sequence of SEQ ID NO:1 and the VLdomain of the anti-IGF-1R antibody comprises the aa sequence of SEQ IDNO:2 or 3. In certain embodiments, the VH domain of an anti-IGF-1Rantibody comprises an aa sequence selected from the group consisting ofSEQ ID NOs: 8-10 and 12-31 and the VL domain comprises an aa sequenceselected from the group consisting of SEQ ID NOs: 32-34 and 36-133.

In certain embodiments, (a) the VH domain of an anti-IGF-1R antibodycomprises an aa sequence that is at least 90% identical to, or whichdiffers in 1-30 aa aa substitutions, additions or deletions from, an aasequence of any of SEQ ID NOs: 8-10 and 12-31, and/or (b) the VL domainof the anti-IGF-1R antibody comprises an aa sequence that is at least90% identical to, or which differs in 1-30 aa aa substitutions,additions or deletions from, an aa sequence of an aa sequence of any ofSEQ ID NOs: 32-34 and 36-133. In certain embodiments, (a) the VH domainof an anti-IGF-1R antibody comprises an aa sequence that is at least 95%identical to, or which differs in 1-10 aa aa substitutions, additions ordeletions from, an aa sequence of any of SEQ ID NOs: 8-10 and 12-31,and/or (b) the VL domain comprises an aa sequence that is at least 95%identical to, or which differs in 1-10 aa aa substitutions, additions ordeletions from, an aa sequence of an aa sequence of any of SEQ ID NOs:32-34 and 36-133.

An anti-IGF-1R antibody may be an IgG1 antibody, e.g., an isolatedand/or a monoclonal IgG1 antibody.

In certain embodiments, an anti-IGF-1R antibody is a protein comprisingtwo pairs of polypeptide chains, each pair comprising a heavy chain anda light chain; wherein (a) each heavy chain comprises an aa sequence ofSEQ ID NOs: 359, 360 or 361; and/or (b) each light chain comprises an aasequence of SEQ ID NOs: 204, 206 or 208. In certain embodiments, ananti-IGF-1R antibody is a protein comprising two pairs of polypeptidechains, each pair comprising a heavy chain and a light chain; wherein(a) each heavy chain comprises an aa sequence that is at least 90%identical to, or which differs in 1-30 aa aa substitutions, additions ordeletions from, an aa sequence of any of SEQ ID NOs:359, 360 or 361,and/or (b) each light chain comprises an aa sequence that is at least90% identical to, or which differs in 1-30 aa aa substitutions,additions or deletions from, an aa sequence of an aa sequence of any ofSEQ ID NOs: 204, 206 or 208. In certain embodiments, (a) each heavychain of an anti-IGF-1R antibody comprises an aa sequence that is atleast 95% identical to, or which differs in 1-10 aa aa substitutions,additions or deletions from, an aa sequence of any of SEQ ID NOs:359,360 or 361, and/or (b) each light chain comprises an aa sequence that isat least 95% identical to, or which differs in 1-10 aa aa substitutions,additions or deletions from, an aa sequence of an aa sequence of any ofSEQ ID NOs: 204, 206 or 208.

Exemplary antibodies include (a) an anti-IGF-R1 monoclonal IgG1 antibodyP4 comprising two heavy chains, each comprising a heavy chain aasequence of SEQ ID NO:359 and two light chains, each comprising a lightchain aa sequence of SEQ ID NO:204; (b) an anti-IGF-R1 monoclonal IgG1antibody M78 comprising two heavy chains, each comprising a heavy chainaa sequence of SEQ ID NO:360 and two light chains, each comprising alight chain aa sequence of SEQ ID NO:206; (c) an anti-IGF-R1 monoclonalIgG1 antibody M57 comprising two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:361 and two light chains, each comprisinga light chain aa sequence of SEQ ID NO:208; (d) an anti-IGF-R1monoclonal IgG1 antibody M57/M78 comprising two heavy chains, eachcomprising a heavy chain aa sequence of SEQ ID NO:361 and two lightchains, each comprising a light chain aa sequence of SEQ ID NO:206; and(e) an anti-IGF-R1 monoclonal IgG1 antibody P4/M57 comprising two heavychains, each comprising a heavy chain aa sequence of SEQ ID NO:359 andtwo light chains, each comprising a light chain aa sequence of SEQ IDNO:208.

Anti-IGF-1R antibodies may comprise one or more additional bindingsites, e.g., an anti-ErbB3 binding site.

Also provided are anti-ErbB3 monoclonal antibodies, e.g., monoclonalanti-ErbB3 antibodies. In certain embodiments, an anti-ErbB3 comprisesin amino to carboxy order a VLCDR1 sequence, a VLCDR2 sequence and aVLCDR3 sequence of P1 lambda light chain as indicated by dottedunderlining in FIG. 5B, SEQ ID NO:258, antibody further comprising inamino to carboxy order a VHCDR1 sequence, a VHCDR2 sequence and a VHCDR3sequence of P1 heavy chain as indicated by the first three dottedunderlined sequences respectively in FIG. 5B, SEQ ID NO:268. In certainembodiments, an anti-ErbB3 monoclonal antibody comprises in amino tocarboxy order a VLCDR1 sequence, a VLCDR2 sequence and a VLCDR3 sequenceof M27 lambda light chain as indicated by dotted underlining in FIG. 5B,SEQ ID NO:260, antibody further comprising in amino to carboxy order aVHCDR1 sequence, a VHCDR2 sequence and a VHCDR3 sequence of M27 heavychain as indicated by the first three dotted underlined sequencesrespectively in FIG. 5B, SEQ ID NO:274. In certain embodiments, ananti-ErbB3 monoclonal antibody comprises in amino to carboxy order aVLCDR1 sequence, a VLCDR2 sequence and a VLCDR3 sequence of M7 lambdalight chain as indicated by dotted underlining in FIG. 5B, SEQ IDNO:262, antibody further comprising in amino to carboxy order a VHCDR1sequence, a VHCDR2 sequence and a VHCDR3 sequence of M7 heavy chain asindicated by the first three dotted underlined sequences respectively inFIG. 5B, SEQ ID NO:280. In certain embodiments, an anti-ErbB3 monoclonalantibody comprises in amino to carboxy order a VLCDR1 sequence, a VLCDR2sequence and a VLCDR3 sequence of B72 lambda light chain as indicated bydotted underlining in FIG. 5B, SEQ ID NO:264, antibody furthercomprising in amino to carboxy order a VHCDR1 sequence, a VHCDR2sequence and a VHCDR3 sequence of B72 heavy chain as indicated by thefirst three dotted underlined sequences respectively in FIG. 5B, SEQ IDNO:286. In certain embodiments, an anti-ErbB3 monoclonal antibodycomprises in amino to carboxy order a VLCDR1 sequence, a VLCDR2 sequenceand a VLCDR3 sequence of B60 lambda light chain as indicated by dottedunderlining in FIG. 5B, SEQ ID NO:266, antibody further comprising inamino to carboxy order a VHCDR1 sequence, a VHCDR2 sequence and a VHCDR3sequence of B60 heavy chain as indicated by the first three dottedunderlined sequences respectively in FIG. 5B, SEQ ID NO:292.

In certain embodiments, an isolated anti-ErbB3 antibody bindingspecifically to human ErbB3 comprises a VH domain comprising a set ofthree VH CDRs comprising VHCDR1, VHCDR2, VHCDR3, and/or a VL domaincomprising a set of three VL CDRs comprising VLCDR1, VLCDR2 and VLCDR3,CDRs comprising the sequences of SEQ ID NOs: 309, 310, 311, 312, 313,and 314, respectively, and each CDR further comprises an amino terminusand a carboxy terminus, wherein the CDRs of each set of CDRs arearranged in the antibody in a linear amino to carboxy order of CDR1,CDR2, and CDR3, wherein the CDRs comprise variable aas, whichindependently represent any aa set forth at the corresponding positionin FIG. 3 (VH) or FIG. 4 (VL), and the antibody does not comprise the C8module. In certain embodiments, the VHCDR1, VHCDR2, VHCDR3, VLCDR1,VLCDR2 and VLCDR3 of an anti-ErbB3 antibody comprise the sequences ofSEQ ID NOs: 309, 310, 311, 312, 313, and 315, respectively. In certainembodiments, the VHCDR1, VHCDR2 and VHCDR3 domains of an anti-ErbB3antibody comprise the corresponding aa sequences of any one of SEQ IDNOs: 134-142 and 144-165, and the VLCDR1, VLCDR2 and VLCDR3 domains ofthe antibody comprise the corresponding aa sequences of any one of SEQID NOs: 166-174 and 176-200.

In certain embodiments, the VHCDR1, VHCDR2, VHCDR3 of an anti-ErbB3antibody comprise the aa sequence of the corresponding CDRs of SEQ IDNO:134 and the VLCDR1, VLCDR2 and VLCDR3 of the anti-ErbB3 antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:166. Incertain embodiments, the VHCDR1, VHCDR2, VHCDR3 of an anti-ErbB3antibody comprise the aa sequence of the corresponding CDRs of SEQ IDNO:135 and the VLCDR1, VLCDR2 and VLCDR3 of the anti-ErbB3 antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:167. Incertain embodiments, the VHCDR1, VHCDR2, VHCDR3 of an anti-ErbB3antibody comprise the aa sequence of the corresponding CDRs of SEQ IDNO:136 and the VLCDR1, VLCDR2 and VLCDR3 of the anti-ErbB3 antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:168. Incertain embodiments, the VHCDR1, VHCDR2, VHCDR3 of an anti-ErbB3antibody comprise the aa sequence of the corresponding CDRs of SEQ IDNO:137 and the VLCDR1, VLCDR2 and VLCDR3 of the anti-ErbB3 antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:169. Incertain embodiments, the VHCDR1, VHCDR2, VHCDR3 of an anti-ErbB3antibody comprise the aa sequence of the corresponding CDRs of SEQ IDNO:138 and the VLCDR1, VLCDR2 and VLCDR3 of the anti-ErbB3 antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:170. Incertain embodiments, the VHCDR1, VHCDR2, VHCDR3 of the anti-ErbB3antibody comprise the aa sequence of the corresponding CDRs of SEQ IDNO:139 and the VLCDR1, VLCDR2 and VLCDR3 of the anti-ErbB3 antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:171. Incertain embodiments, the VHCDR1, VHCDR2, VHCDR3 of an anti-ErbB3antibody comprise the aa sequence of the corresponding CDRs of SEQ IDNO:140 and the VLCDR1, VLCDR2 and VLCDR3 of the antibody comprise the aasequence of the corresponding CDRs of SEQ ID NO:172. In certainembodiments, the VHCDR1, VHCDR2, VHCDR3 of an anti-ErbB3 antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:141 andthe VLCDR1, VLCDR2 and VLCDR3 of the anti-ErbB3 antibody comprise the aasequence of the corresponding CDRs of SEQ ID NOs: 173. In certainembodiments, the VHCDR1, VHCDR2, VHCDR3 of an anti-ErbB3 antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:142 andthe VLCDR1, VLCDR2 and VLCDR3 of the anti-ErbB3 antibody comprise the aasequence of the corresponding CDRs of SEQ ID NO:174. In certainembodiments, the VHCDR1, VHCDR2, VHCDR3 of an anti-ErbB3 antibodycomprise the aa sequence of the corresponding CDRs of SEQ ID NO:136 andthe VLCDR1, VLCDR2 and VLCDR3 of the anti-ErbB3 antibody comprise the aasequence of the corresponding CDRs of SEQ ID NO:169.

In certain embodiments, the VH domain of an anti-ErbB3 antibodycomprises the aa sequence of SEQ ID NO:4 (or 5), which sequencecomprises variable aas, which independently represent any aa set forthat the corresponding position in FIG. 3 and/or the VL domain of theanti-ErbB3 antibody comprises the aa sequence of SEQ ID NO:6 (or 7),which sequence comprises variable aas, which independently represent anyaa set forth at the corresponding position in FIG. 4. In certainembodiments, The VH domain of an anti-ErbB3 antibody comprises an aasequence selected from the group consisting of SEQ ID NOs: 134-142 and144-165 and/or the VL domain of the anti-ErbB3 antibody comprises an aasequence selected from the group consisting of SEQ ID NOs: 166-174 and176-200.

In certain embodiments, (a) the VH domain of an anti-ErbB3 antibodycomprises an aa sequence that is at least 90% identical to, or whichdiffers in 1-30 aa aa substitutions, additions or deletions from, an aasequence of any of SEQ ID NOs: 134-142 and 144-165, and/or (b) the VLdomain of the anti-ErbB3 antibody comprises an aa sequence that is atleast 90% identical to, or which differs in 1-30 aa aa substitutions,additions or deletions from, an aa sequence of an aa sequence of any ofSEQ ID NOs: 166-174 and 176-200. In certain embodiments, (a) the VHdomain of an anti-ErbB3 antibody comprises an aa sequence that is atleast 95% identical to, or which differs in 1-10 aa aa substitutions,additions or deletions from, an aa sequence of any of SEQ ID NOs:134-142 and 144-165, and/or (b) the VL domain comprises an aa sequencethat is at least 95% identical to, or which differs in 1-10 aa aasubstitutions, additions or deletions from, an aa sequence of an aasequence of any of SEQ ID NOs: 166-174 and 176-200.

An anti-ErbB3 antibody may be an IgG1 antibody, e.g., an isolatedmonoclonal IgG1 antibody.

In certain embodiments, an anti-ErbB3 antibody is a protein comprisingtwo pairs of polypeptide chains, each pair comprising a heavy chain anda light chain; wherein (a) each heavy chain comprises an aa sequence ofSEQ ID NOs: 362, 363, 364, 365 or 366; and/or (b) each light chaincomprises an aa sequence of SEQ ID NOs: 258, 260, 262, 264 or 266. Incertain embodiments, an anti-ErbB3 antibody is a protein comprising twopairs of polypeptide chains, each pair comprising a heavy chain and alight chain; wherein (a) each heavy chain comprises an aa sequence thatis at least 90% identical to, or which differs in 1-30 aa aasubstitutions, additions or deletions from, an aa sequence of any of SEQID NOs: 362, 363, 364, 365 or 366, and/or (b) each light chain comprisesan aa sequence that is at least 90% identical to, or which differs in1-30 aa aa substitutions, additions or deletions from, an aa sequence ofan aa sequence of any of SEQ ID NOs: 258, 260, 262, 264 or 266. Incertain embodiments, an anti-ErbB3 antibody is a protein comprising twopairs of polypeptide chains, each pair comprising a heavy chain and alight chain; wherein (a) each heavy chain comprises an aa sequence thatis at least 95% identical to, or which differs in 1-10 aa aasubstitutions, additions or deletions from, an aa sequence of any of SEQID NOs: 362, 363, 364, 365 or 366, and (b) each light chain comprises anaa sequence that is at least 95% identical to, or which differs in 1-10aa aa substitutions, additions or deletions from, an aa sequence of anaa sequence of any of SEQ ID NOs: 258, 260, 262, 264 or 266.

An anti-ErbB3 antibody may comprise one or more other binding sites,e.g., an anti-IGF-1R binding site.

Exemplary anti-ErbB3 antibodies include: (a) an anti-ErbB3 monoclonalIgG1 antibody P1 comprising two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:362 and two light chains, each comprisinga light chain aa sequence of SEQ ID NO:258; (b) an anti-ErbB3 monoclonalIgG1 antibody M27 comprising two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:363 and two light chains, each comprisinga light chain aa sequence of SEQ ID NO:260; (c) an anti-ErbB3 monoclonalIgG1 antibody M7 comprising two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:364 and two light chains, each comprisinga light chain aa sequence of SEQ ID NO:262; (d) an anti-ErbB3 monoclonalIgG1 antibody B72 comprising two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:365 and two light chains, each comprisinga light chain aa sequence of SEQ ID NO:264; (e) an anti-ErbB3 monoclonalIgG1 antibody B60 comprising two heavy chains, each comprising a heavychain aa sequence of SEQ ID NO:366 and two light chains, each comprisinga light chain aa sequence of SEQ ID NO:266; and (f) an anti-ErbB3monoclonal IgG1 antibody M27/M7 comprising two heavy chains, eachcomprising a heavy chain aa sequence of SEQ ID NO:363 and two lightchains, each comprising a light chain aa sequence of SEQ ID NO:262.

Also provided are scFvs, which may be monoclonal scFvs. Exemplary scFvsinclude (a) anti-IGF-R1 scFv antibody P4 comprising an aa sequence ofSEQ ID NO:367; (b) anti-IGF-R1 scFv antibody M57 comprising an aasequence of SEQ ID NO:368; (c) anti-IGF-R1 scFv antibody M78 comprisingan aa sequence of SEQ ID NO:369; (d) anti-ErbB3 scFv antibody C8comprising an aa sequence of SEQ ID NO:370; (e) anti-ErbB3 scFv antibodyP1 comprising an aa sequence of SEQ ID NO:371; (f) anti-ErbB3 scFvantibody M1.3 comprising an aa sequence of SEQ ID NO:372; (g) anti-ErbB3scFv antibody M27 comprising an aa sequence of SEQ ID NO:373; (h)anti-ErbB3 scFv antibody P6 comprising an aa sequence of SEQ ID NO:374;and (i) anti-ErbB3 scFv antibody B69 comprising an aa sequence of SEQ IDNO:375.

Also provided are compositions comprising a PBA; an anti-IGF-1Rantibody; or an anti-ErbB3 antibody and a pharmaceutically acceptablecarrier. A composition comprising an anti-IGF-1R antibody may furthercomprise an anti-ErbB3 antibody. A composition comprising an anti-ErbB3antibody may further comprise an anti-IGF-1R antibody.

Also provided are nucleic acid molecules, e.g., comprising at least onecoding sequence, at least one coding sequence encoding an antibody or achain thereof, as set forth herein. The nucleic acid molecule maycomprise either or both of a promoter nucleotide sequence and anenhancer nucleotide sequence, which nucleotide sequence is operablylinked to the at least one coding sequence and promotes or enhances theexpression of the antibody. Also encompassed are vectors, e.g., vectorscomprising one or more nucleic acid molecules provided herein, as wellas host cells comprising one or more vectors provided herein. Alsoprovided are methods for producing a PBA, an anti-IGF-1 antibody or ananti-ErbB3 antibody provided herein, comprising culturing a cellcomprising one or more nucleic acids encoding the antibodies (e.g., aPBA) or chains thereof provided herein under conditions suitable for theexpression of the PBA, anti-IGF-1 antibody or anti-ErbB3 antibody.Further provided are methods for treating a subject having cancer, saidmethod comprising administering to the subject a therapeuticallyeffective amount of one or more antibodies or PBAs or compositionsprovided herein.

Also provided here are anti-IGF-1R+anti-ErbB3 PBAs, wherein the PBA hasa half-life of at least 45 hours in a Cynomolgus monkey, when the PBA isadministered intravenously at doses equal to or higher than 5 mg/kg. Incertain embodiments, a PBA has a half-life that is statisticallysignificantly longer (e.g., by 50%, 2 fold or more) in an organism thatis a mouse or a cynomolgus monkey than the half-life of anotherpolyvalent bispecific ab in the same organism, binding to the sameepitopes, wherein the orientation of antigen binding specificities isreversed between of the fab and of the scfv.

PBAs may suppress heregulin-induced pAKT signaling in a cell, e.g., acancer cel, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100%. PBAs may suppress IGF-1-inducedpAKT signaling in a cell, e.g., a cancer cell, by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%.PBAs may suppress insulin-induced pAKT signaling in a cell, e.g., acancer cell, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100%. PBAs may suppress theIGF-2-induced pAKT signaling in a cell, e.g., a cancer cell, by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%%, 97%, 98%,99% or 100%.

A PBA may inhibit mTOR activation (phosphorylation) in a tumor cell invivo or in vitro to a greater extent than monospecific anti-IGF-1R Ab# Aor than a monospecific anti-IGF-1R Ab that binds to the same epitope onIGF-1R as the PBA. PBAs may reduce mTOR protein levels in a tumor cellin vivo or in vitro to a greater extent than monospecific anti-IGF-1RAb# A, or than a monospecific anti-IGF-1R Ab that binds to the sameepitope on IGF-1R as the PBA. In certain embodiments, a PBA reduces mTORactivation or mTOR protein levels in a tumor cell in vivo or in vitro bya factor of at least 50%, or by 2, 3, 4 or 5 fold or greater than 5 foldrelative to monospecific anti-IGF-1R Ab# A or relative to a monospecificanti-IGF-1R Ab that binds to the same epitope on IGF-1R as the PBA. Incertain embodiments, a PBA is more effective at inhibiting tumor growthin a human xenograft model in nu/nu mice than is an equimolar amount ofan anti-IGF-1R IgG. In some embodiments the xenograft models comprisehuman DU145, BxPC-3, SK-ES-1, or Caki-1 cell xenografts. In anotherembodiment, a polyvalent bispecific antibody is more effective atinhibiting tumor growth in a human xenograft model in nu/nu mice than isa combination of an equimolar amount of an anti-IGF-1R IgG combined withan equimolar amount of an anti-ErbB3 IgG.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B: an alignment of exemplary IGF-1R VH sequences (SEQ IDNOs:8-31 and 384-385, numbered consecutively from top to bottom) and aconsensus sequence (SEQ ID NO:1) derived therefrom. The CDRs areunderlined and the SEQ ID NOs of the CDRs are provided above the CDRs asa number in square brackets (e.g., “[S.302]”).

FIG. 2A-E: an alignment of exemplary IGF-1R VL sequences (SEQ IDNOs:32-133 and 386-387, numbered consecutively from top to bottom) andtwo consensus sequences (SEQ ID NOs:2 and 3) derived therefrom. SEQ IDNO:2 includes the VL domain of 16F, whereas SEQ ID NO:3 does not. TheCDRs are underlined and the SEQ ID NOs of the CDRs are provided abovethe CDRs as a number in square brackets.

FIG. 3A-B: an alignment of exemplary ErbB3 VH sequences (SEQ IDNOs:134-165 and 388, numbered consecutively from top to bottom) and twoconsensus sequences (SEQ ID NOs:4 and 5) derived therefrom. SEQ ID NO:4includes the VH domain of 16F, whereas SEQ ID NO:5 does not. The CDRsare underlined and the SEQ ID NOs of the CDRs are provided above theCDRs as a number in square brackets.

FIG. 4A-B: an alignment of exemplary ErbB3 VL sequences (SEQ IDNOs:166-200, numbered consecutively from top to bottom) and twoconsensus sequences (SEQ ID NOs:6 and 7) derived therefrom. SEQ ID NO:6includes the VL domain of 16F, whereas SEQ ID NO:7 does not. The CDRsare underlined and the SEQ ID NOs of the CDRs are provided above theCDRs as a number in parenthesis.

FIG. 5A-B: Aa sequences of the light and heavy chains of exemplaryanti-IGF-1R-IgG1-anti-ErbB3 (FIG. 5 A) and anti-ErbB3-IgG1-anti-IGF-1R(FIG. 5B) polyvalent bispecific antibodies.

FIG. 5A shows the aa sequences of the followinganti-IGF-1R-IgG1-anti-ErbB3 hybrid heavy chains: SF-G1-C8 (i.e., 16F—SEQID NO:210); SF-G1-P1 (SEQ ID NO:212); SF-G1-M1.3 (SEQ ID NO:214);SF-G1-M27 (SEQ ID NO:216); SF-G1-P6 (SEQ ID NO:218); SF-G1-B69 (SEQ IDNO:220); P4-G1-C8 (SEQ ID NO:222); P4-G1-P1 (SEQ ID NO:224); P4-G1-M1.3(SEQ ID NO:226); P4-G1-M27 (SEQ ID NO:228); P4-G1-P6 (SEQ ID NO:230);P4-G1-B69 (SEQ ID NO:232); M78-G1-C8 (SEQ ID NO:234); M78-G1-P1 (SEQ IDNO:236); M78-G1-M1.3 (SEQ ID NO:238); M78-G1-M27 (SEQ ID NO:240);M78-G1-P6 (SEQ ID NO:242); M78-G1-B69 (SEQ ID NO:244); M57-G1-C8 (SEQ IDNO:246); M57-G1-P1 (SEQ ID NO:248); M57-G1-M1.3 (SEQ ID NO:250);M57-G1-M27 (SEQ ID NO:252); M57-G1-P6 (SEQ ID NO:254) and M57-G1-B69(SEQ ID NO:256).

FIG. 5B shows the aa sequences of the followinganti-ErbB3-IgG1-anti-IGF-1R hybrid heavy chains: P1-G1-P4 (SEQ IDNO:268); P1-G1-M57 (SEQ ID NO:270); P1-G1-M78 (SEQ ID NO:272); M27-G1-P4(SEQ ID NO:274); M27-G1-M57 (SEQ ID NO:276); M27-G1-M78 (SEQ ID NO:278);M7-G1-P4 (SEQ ID NO:280); M7-G1-M57 ((SEQ ID NO:282); M7-G1-M78 (SEQ IDNO:284); B72-G1-P4 (SEQ ID NO:286); B72-G1-M57 (SEQ ID NO:288);B72-G1-M78 (SEQ ID NO:290); B60-G1-P4 (SEQ ID NO:292); B60-G1-M57 (SEQID NO:294); B60-G1-M78 (SEQ ID NO:296); B60-G2-M78 (SEQ ID NO:355) andM7-G2-M78 (SEQ ID NO:357).

Each of these hybrid heavy chains of FIGS. 5A and 5B comprises threenamed modules and each is named according to its modular composition(see FIG. 8), with, on the left, the name of a first, amino-terminalmodule, in the middle, the name of the second, middle module (always G1or G2, as indicated, in the polyvalent bispecific antibodies whosesequences are provided in FIGS. 5A and 5B) and on the right, the name ofthe third, carboxy-terminal module. Each first, amino-terminal modulecomprises a heavy chain VR comprising from left to right a VHCDR1, aVHCDR2 and a VHCDR3, each indicated by dotted underlining. Each second,middle module is named G1 or G2 and comprises an IgG1 CR, and does notform an antigen binding site in a polyvalent bispecific antibody. Thehinge, CH2 and CH3 portions of the G1 or G2 module sequence is singleunderlined. The CH1 portion starts at ASTK (SEQ ID NO:392). A Gly-Serlinker sequence linking the G1 module with the third module is doubleunderlined. Each third, carboxy-terminal module appears to the right ofthis Gly-Ser linker sequence and comprises an scFv that comprises aheavy chain VR comprising from left to right a VHCDR1, a VHCDR2 and aVHCDR3 (each dotted underlined), a Gly-Ser scFv linker (double wavyunderlined), and a light chain VR comprising a VLCDR1, a VLCDR2 and aVLCDR3 (each dotted underlined).

The binding specificity of each amino or carboxy-terminal heavy chainmodule is the same as the correspondingly named light chain of FIG. 5Aor 5B.

FIG. 5A shows aa (“aa”) sequences of the following mature anti-IGF-R1kappa light chains: SF (SEQ ID NO:202), P4 (SEQ ID NO:204), M78 (SEQ IDNO:206), and M57 (SEQ ID NO:208).

FIG. 5B shows aa sequences of the following mature anti-ErbB3 lambdalight chains: P1 (SEQ ID NO:258), M27 (SEQ ID NO:260), M7 (SEQ IDNO:262), B72 (SEQ ID NO:264), and B60 (SEQ ID NO:266).

Each light chain of FIGS. 5A and 5B comprises, from left to right, aVLCDR1, a VLCDR2 and a VLCDR3 (each dotted underlined). The CL domainstarts at “RTVAA” (SEQ ID NO:393) in the anti-IGF-1R VL domain and at“QPKAA” (SEQ ID NO:394) in the anti-ErbB3 VL domain.

To form an entire polyvalent bispecific antibody comprising the heavyand light chains of FIGS. 5A and 5B, each heavy chain is co-expressedwith a light chain which shares the name of the amino-terminal module ofthe heavy chain. Each of the resultant polyvalent bispecific antibodiestakes the form of an IgG antibody (which comprises, as do native IgGantibodies, two essentially identical antigen binding sites) with anscFv appended to the carboxy terminus of each of the two heavy chains ofthe IgG.

FIG. 6A-B: Aa sequences of the heavy chains of exemplary 6A) anti-IGF-1RIgG1 antibodies and 6B) anti-ErbB3 IgG1 antibodies; and aa sequences of6C) anti-IGF-1R scFvs, and 6D) anti-ErbB3 scFvs.

FIG. 7A-B: Aa sequences of 7A) the heavy chains and 7B) the lightchains, of SF-G1-C8 (16F), each with a leader sequence that is absent inthe mature antibody. The heavy chain aa sequence (SEQ ID NO:300) is thatof SF-G1-C8 (SEQ ID NO:210) with an added N-terminal leader sequence.The light chain aa sequence (SEQ ID NO:298) is that of SF kappa lightchain (SEQ ID NO:202) with an added N-terminal leader sequence. Theleader sequences are in boldface and underlined; the dotted underlinedsequences are CDRs as indicated above each; the linkers (hinge,connecting linker and scFv linker) are indicated above their boldfacedsequences, and the individual CH3 aa residues E356 and M358 are in bold(these are aas that can be substituted as follows E356D and M358L). Theidentities of the CH1, CH2, CH3, VH and VL domains in FIG. 7A and the CLdomain in FIG. 7B are indicated above each domain, with the start pointof each indicated by a right-angled arrow.

FIG. 8: Schematic view showing the derivation of modules of an Ig-liketetravalent bispecific antibody. The antibodies from which the bindingdomains (“N-terminal module” and “C-terminal module”) are derived arelabeled monoclonal antibody 1 and monoclonal antibody 2. The diagramsand modules are conceptual in nature; the actual DNA fragments that arejoined together to prepare such a bispecific antibody may not coincidewith the limits of the modules, but the end result is essentially asshown. Furthermore, monoclonal antibody 1 and monoclonal antibody 2 maynot be in IgG format as shown—for example, either or both may be anscFv. If an scFv is used as the source of an N-terminal module, it isconverted from scFv format to Fv format by removal of the DNA sequenceencoding the scFv linker to yield VH and VL regions that are comprisedby heavy and light polypeptide chains respectively.

FIG. 9A-D: Simulation of the ErbB network predicts design of an optimalErbB3 therapeutic. 9A) Complexity of the ErbB network depictedgraphically: Each of ligand binding, receptor dimerization, receptortrafficking and intracellular signaling were captured in a mass-actionbased kinetic model. 9B) Simulated perturbation of each protein in theErbB network was used to identify the sensitivity of the downstreamsignal, phospho-Akt, towards each protein under either heregulin orbetacellulin stimulation. Dose responsiveness of Akt to an anti-ErbB3antibody under either heregulin (9C) or betacellulin (9D) stimulationwas examined for a variety of affinity binding constants throughvariation of the dissociation rate. The order of K_(d)s listed for eachgraph from top to bottom corresponds to the order of curves in eachgraph from left to right.

FIG. 10A-C: Ability of a bispecific antibody to co-inhibit two pathwaysis predicted to be dependent on relative receptor levels. The effect ofa computationally simulated bispecific antibody on the active level ofA) IGF-1R, B) ErbB3 and C) a common downstream signaling pathwayelement, Akt (the Akt kinase), was computationally simulated for cellsexpressing three different molar ratios of each receptor to the other.The three curves in each graph can be identified by the relativepositions at which each of their right-hand ends intersects the boarderof the graph. Leftmost x-axis/bottommost y-axis intersectingcurve=simulation of 10 times more of IGF-1R than ErbB3, middleintersecting curve=simulation of equal levels of IGF-1R and ErbB3,rightmost x-axis/topmost y-axis intersecting curve=simulation of 10times more ErbB3 than IGF-1R. Due to the lower affinity of thebispecific towards IGF-1R, potency of IGF-1R inhibition decreases whenthe ErbB3 levels decrease, indicating the effect of avidity. Failure topotently inhibit IGF-1R leads to a decreased ability to inhibit pAkt(right). The ability to inhibit ErbB3 is unaffected by the level ofIGF-1R due the stronger affinity of the bispecific towards ErbB3.

FIG. 11A-B: Concurrent optimization of scFv affinity and stability bycovalent yeast display. 11A) Affinity of post-thermal-challenge-isolatedyeast-displayed scFv modules to soluble GFR2(ErbB3)-Fc measured byfluorescence-activated cell sorting. 11B) Thermal challenge assay onoptimized scFv modules covalently attached to yeast surface confirmstheir higher thermal stability. The residual binding activity toErbB3-Fc remaining after heat stress for 5 minutes at 65° C. wasmeasured by fluorescence-activated cell sorting. MFI=mean fluorescenceintensity.

FIG. 12A-B: Differential scanning fluorescence can be used to estimateserum stability. 12A) Microscopic stability measurement by differentialscanning fluorimetry. 12B) Macroscopic stability measured by percent ofbinding activity before (100%) and after 3 day incubation in mouse serumat 37° C. The proof-of-concept bispecific protein exhibits inferiorstability compared to a stabilized analog in both microscopic (A) andmacroscopic (B) properties.

FIG. 13: Optimized bispecific antibody displays stronger cellularbinding compared to proof-of-concept bispecific antibody. Binding toBxPC-3 cells, which express both IGF-1R and ErbB3, was measured byfluorescence-activated cell sorting.

FIG. 14A-B: Binding to ADRr and MCF7 cells. 14A) Binding to ADRr cells.As used in this figure and in FIGS. 14B, 18, 19, 20A and 20B, below,“IgG of module 2-21” refers to an anti-ErbB3 antibody having the sameanti-ErbB3 VRs as those in ILE-12. “IgG of module 2-3” refers ananti-ErbB3 antibody having the same anti-ErbB3 VRs as those in ELI-7 andILE-10. “IgG of module 5-7” refers to an anti-IGF-1R antibody having thesame anti-IGF-1R VRs as those in ELI-7, ILE-10 and ILE-12. 14B) Bindingto MC7 cells.

FIG. 15: pAKT inhibition by ILE-7 and ELI-7.

FIG. 16: Effect of ELI-7 on growth of DU145 cells (measured by CTGassay). RLU=relative luminescence units.

FIG. 17: Inhibition of BxPC-3 cell growth by ELI-7 measured by CTGassay.

FIG. 18: Xenograft tumor growth curves. A description of the IgGantibodies is provided in the figure legend of FIG. 14A.

FIG. 19: BxPC-3 final xenograft tumor volumes on Day 41.

FIG. 20A-B: Xenograft tumor growth rates and sizes. 20A) DU145 TumorGrowth Curves. 20B) DU145 Tumor Volumes on Day 36.

FIG. 21: Polyvalent bispecific antibodies inhibit signaling across abroad range of ErbB3 and IGF-1R receptor levels. ELI-7 displaysinhibition of pAkt across BxPC-3 cell lines modified to contain a broadrange of IGF1R and ErbB3 receptor levels. “BxPC-3-Control” refers toBxPC-3 cells with unchanged IGF-1R and ErbB3 levels. “BxPC-3-IGF1R-Mod1”refers to BxPC-3 cells in which the IGF-1R level is reduced by 37%.“BxPC-3-ErbB3-Mod1” refers to BxPC-3 cells in which the ErbB3 level isreduced by 48%. “BxPC-3-ErbB3-Mod2” refers to BxPC-3 cells in which theErbB3 level is reduced by 88%.

FIG. 22: Reduction of pIGF-1R levels by 16F (SF-G1-C8) “re-engineeredbispecific” and ELI-7 “original bispecific” in BcPC3 cells.

FIG. 23A-C: Inhibition by 16F (SF-G1-C8), Anti-IGF-1R Ab#B (cixutumumab;SEQ ID 324+SEQ ID 325), Anti-ErbB3 Ab# A (SEQ ID NO:336+SEQ ID NO:337)or Anti-IGF-1R Ab#B+Anti-ErbB3 Ab# A of phosphorylation of: 23A) IGF1R,23B) ErbB3, and 23C) AKT, in BxPC-3 cells and inhibition by 16F(SF-G1-C8), Anti-IGF-1R Ab#B (cixutumumab; SEQ ID NO:324+SEQ ID NO:325),Anti-ErbB3 Ab# A (SEQ ID NO:336+SEQ ID NO:337) or Anti-IGF-1RAb#B+Anti-ErbB3 Ab# A of phosphorylation of: 23D) IGF1R, 23E) ErbB3, and23F) AKT, in DU145 cells.

FIGS. 24 A-F: Signaling inhibition by 16F (SF-G1-C8) (FIGS. 24A-C)compared to ANTI-IGF-1R Ab# A (ganitumab; SEQ ID NO:327+SEQ ID NO:328),anti-ErbB3 Ab# A (SEQ ID NO:336+SEQ ID NO:337) and ANTI-IGF-1R Ab#A+anti-ErbB3 Ab# A (FIGS. 24D-F). Inhibition of phosphorylation of IGF1Ris shown in the top graph (FIGS. 24A and D), inhibition ofphosphorylation of ErbB3 is shown in the middle graph (FIGS. 24B and E),and inhibition of phosphorylation of AKT is shown in the bottom graph(FIGS. 24C and F); all in BxPC-3 cells.

FIG. 25: Bispecific antibodies display strong binding to BxPC-3 cells.Binding curves generated after incubation of indicated antibodies withBxPC-3 cells as measured by FACS.

FIG. 26: Bispecific antibodies display strong binding to recombinantErbB3 protein, Binding curves generated after incubation of indicatedantibodies in ErbB3-His-coated plates and measurement of bound antibodylevels by ELISA.

FIGS. 27 A-C: Bispecific antibodies display strong inhibition of dualpathway signaling. BxPC-3 signal inhibition of generation of pIGF1R(FIG. 27A), pErbB3 (FIG. 27B), and pAKT (FIG. 27C), as indicated.

FIG. 28: Percent stability of indicated bispecific antibodies in serumfor 72 hrs at 37° C.

FIG. 29: A-D show the binding of various bispecific antibodies (asindicated) to BxPC-3 cells as measured by FACS. In 29A), the N-terminalmodules of M27/M7-IgG-P4, M27/M7-IgG-M57, and M27/M7-IgG-M78 bispecificantibodies contain the M27 heavy chain and the M7 light chain.

FIGS. 30 A-I: show BxPC-3 signal inhibition data for various bispecificantibodies (as indicated) as measured by changes in pIGF1R levels.

FIGS. 31 A-K: show BxPC-3 signal inhibition data for various bispecificantibodies (as indicated) as measured by changes in pErbB3 levels.

FIGS. 32 A-I: show BxPC-3 signal inhibition data for various bispecificantibodies (as indicated) as measured by changes in pAKT levels.

FIG. 33A-D: show BxPC-3 signal inhibition data for various bispecificantibodies (as indicated) compared to a combination of anti-ErbB3 Ab# A(SEQ ID NO:336+SEQ ID NO:337) and ANTI-IGF-1R Ab# A (ganitumab; SEQ IDNO:327+SEQ ID NO:328), as measured by changes in pIGF1R levels.

FIG. 34 A-D: show BxPC-3 signal inhibition data for various bispecificantibodies (as indicated) compared to a combination of anti-ErbB3 Ab# A(SEQ ID NO:336+SEQ ID NO:337) and ANTI-IGF-1R Ab# A (ganitumab; SEQ IDNO:327+SEQ ID NO:328), as measured by changes in pErbB3 levels.

FIG. 35 A-D: show BxPC-3 signal inhibition data for various bispecificantibodies compared to a combination of anti-ErbB3 Ab# A (SEQ IDNO:336+SEQ ID NO:337) and ANTI-IGF-1R Ab# A (ganitumab; SEQ IDNO:327+SEQ ID NO:328), as measured by changes in pAKT levels.

FIG. 36 A-B: show normalized stability for various bispecific antibodies(as indicated) in mouse serum for 5 days at 37° C.

FIGS. 37 A-C: Published aa sequences of heavy chains, light chains andscFvs of anti-IGF-1R antibodies that may be incorporated into polyvalentbispecific antibodies in accordance with the disclosure herein.

FIGS. 38 A-D: Published aa sequences of heavy chains, light chains andscFvs of anti-ErbB3 antibodies to be incorporated into polyvalentbispecific antibodies in accordance with the disclosure herein.

FIG. 39 A-D: A-B show inhibition of IGF1 and heregulin (HRG) signaltransduction in DU145 cells by the PBAs A) M7-G1-M78 (“M7-M78”),P4-G1-M1.3 (“P4-M1.3”), P4-G1-C8 (“P4-C8”) and B) SF-G1-C8 (“SF-C8”)compared to the absence of PBAs (“IGF1+HRG”); the absence of inducer andPBA (“No Tx”); anti-IGF1R mAB alone; anti-ErbB3 mAb alone; and acombination of anti-IGF-1R+anti-ErbB3, as measured by inhibition ofphosphorylation of AKT. C-D show inhibition data obtained similarly tothat in A-B, but in BxPC-3 cells. Anti-IGF1R and anti-ErbB3 mAbs in thisFig. and in FIGS. 40-44 and 51 are ANTI-IGF-1R Ab# A (ganitumab; SEQ IDNO:327+SEQ ID NO:328) and anti-ErbB3 Ab# A (SEQ ID NO:336+SEQ IDNO:337), respectively.

FIG. 40 A-F: show inhibition of IGF1 and heregulin (HRG) induced signaltransduction by the PBAs M7-G1-M78, P4-M 1.3, P4-C8 and SF-C8 in BxPC-3cells which have A-D) wild type levels of IGF-1R and ErbB3; B-E) levelsof IGF-1R reduced by about 50%; or C-F) levels of ErbB3 reduced by about50%, as measured by inhibition of phosphorylation of AKT.

FIG. 41 A-D: show inhibition of signal transduction induced by A-B) 40ng/ml of IGF1 or C-D) or 400 ng/ml IGF1 by the PBAs M7-G1-M78, P4-M1.3,P4-C8 and SF-C8 in BxPC-3 cells, as measured by inhibition ofphosphorylation of AKT.

FIG. 42 A-D: show inhibition of signal transduction induced by A-B) 20ng/ml of IGF1 or C-D) or 200 ng/ml heregulin (HRG) by the PBAsM7-G1-M78, P4-M1.3, P4-C8 and SF-C8 in BxPC-3 cells, as measured byinhibition of phosphorylation of AKT.

FIG. 43A-D: show inhibition of basal signaling in A549 cells after A) 15minutes incubation with the PBAs M7-G1-M78, P4-M1.3, P4-C8 and SF-C8; orB) 24 hours incubation with the PBAs. C-D show inhibition of basalsignaling in BsPC-3 cells after C) 15 minutes incubation with the PBAsM7-G1-M78, P4-M1.3, P4-C8 and SF-C8; or D) 24 hours incubation with thePBAs. All signaling is determined by measuring pAKT levels.

FIGS. 44 A-B: show the total IGF1R level in A549 cells (FIG. 44A) andBxPC-3 cells (FIG. 44B), after 24 hours of treatment with P4-G1-C3 orP4-G1-M1.3.

FIG. 45: Western blot showing the protein level of pIGF1R, pAKt, andB-Actin in DU145 or MIA PaCa-2 (High IR) cells that were serum starved(“Starved”) or treated with IGF1 or IGF2 alone or in the presence of thePBA P4-M1.3 or P4-C8.

FIG. 46: Western blot showing the protein level of pIGF1R, pAKt, andB-Actin in DU145 cells that were serum starved (“Starved”) or treatedwith IGF1; IGF1 in the presence of P4-M1.3; insulin or insulin in thepresence of P4-M1.3.

FIG. 47: Western blot showing the protein level of IGF1R, pIGF1R, ErbB3,pErbB3, pAkt and B-Actin in BxPC-3 cells incubated in the absence ofligand (lanes 1-3) or in the presence of IGF1 and heregulin (HRG) (lanes4-6) and in the absence of a PBA or in the presence of PBA M7-78 orP4-C8.

FIG. 48 A-F: show the amount of A-B) M7-G1-M78; C-D) P4-G1-M1.3; E-F)P4-G1-C8 present after 0 or 5 days incubation in mouse or Cynomolgusmonkey serum. G-H show the amount of P4G1-M1.3 present after 0 or 6 daysin human serum, in plates coated with IGF-1R (G) or ErbB3 (H).

FIG. 49 A-F: show the level of binding to A-C) human, mouse, rat andCynomolgus ErbB3 and D-F) human, mouse, rat and Cynomolgus EGF-1R ofPBAs P4-C8 (A, D), P4-M1.3 (B, E) and M7-M78 (C, F). Binding of M7-M78to rat and Cynomolgus IGF-1R is not provided.

FIG. 50: A-B show the concentration of PBA P4-G1-M1.3 that is necessaryto detach A) IGF1 and B) IGF2 from IGF-1R bound to a plate.

FIG. 51 A-F: show ng per total mg protein of Phospho-IGF-1R (A),Phospho-ErbB3 (B), Phospho-Akt (C), Phospho-ERK (p44/p42; D),Phospho-mTOR (Ser2448, E), and Phospho-S6 (Ser235/236; 5F) in vitro overtime in BxPC-3 cells treated with IGF-1+HRG or IGF-1+HRG+P4-G1-M1.3.

FIG. 52: Levels of pIGF1R, pAKT and B actin in BxPC-3 and A673 cellstreated with serum, IGF1, IGF1 and P4M1.3 (P4-G1-M1.3), IGF2, IGF2 andP4M1.3 (P4-G1-M1.3), insulin, and insulin and P4M1.3 (P4-G1-M1.3).

FIG. 53: A-B show the level of mTOR (A) and phospho-mTOR (“pmTOR”) inend of study BxPC-3 tumors of mice in which one of PBS, P4-G1-M1.3 oranti-IGF-1R Ab# A was injected.

FIGS. 54 A-K: show the level of IGF-1R (A) and insulin receptor (B),ErbB3 (C) and EGFR (D), AKT phosphorylated on residue S473 (“pAKT S473”)(E) and T308 (“pAKT T308”) (F), phospho-Fox01 and Fox03a, “Phospho-Fox01(Thr24)/Fox03a (Thr32”)), mTOR phosphorylated on residue S2448 (H) andS2481 (I), and S6 phosphorylated on residue S235/236 (J) and S 240/244(K), “pS6 S235/236” and “pS6 S240/244”)) in end of study Caki-1 tumorsof mice in which one of PBS, P4-G1-M1.3, anti-IGF-1R Ab# A+anti-ErbB3IgG, and the mTOR inhibitor everolimus was injected.

FIGS. 55 A-E: show the level of IGF-1R (A) and ErbB3 (B), phospho-EGFR(“pEGFR”) (C), and phospho-mTOR (“pmTOR S2448”) (D) and phospho-S6 (“pS6S235/236”) (E) in end of study BxPC-3 tumors of mice in which one ofPBS, P4-G1-M1.3, and anti-IGF-1R Ab# A was injected.

FIGS. 56 A-B: shows the level of IGF-1 (“IGF1”, left panel) (A) andIGF-2 (“IGF2”, right panel) (B) in an ELISA assay wherein plates werecoated with IGF-1R-His and a serial dilution of P4-G1-M1.3 was added tothe wells.

FIG. 57 A-D: shows the mean tumor volume over time in a DU145 (A),BxPC-3 (B), SK-ES-1 (C), and Caki-1 (D) xenograft model. Mice wereinjected with one of PBS, 500 μg P4-G1-M1.3, 100 μg P4-G1-M1.3, 500 μgP4-G1-C8, or 100 μg P4-G1-C8 (A); PBS, 500 μg P4-G1-M1.3, 300 μgP4-G1-M1.3, 100 μg P4-G1-M1.3, 500 μg P4-G1-C8, 300 μg P4-G1-C8, 100 μgP4-G1-C8, 375 μg anti-IGF-1R Ab# A (ganitumab; SEQ ID NO:327+SEQ IDNO:328), 225 μg anti-IGF-1R Ab# A (ganitumab; SEQ ID NO:327+SEQ IDNO:328), or 75 μg anti-IGF-1R Ab# A (ganitumab; SEQ ID NO:327+SEQ IDNO:328) (B); PBS, 500 μg P4-G1-M1.3, 300 μg P4-G1-M1.3, or 100 μgP4-G1-M1.3 (C); or PBS, 500 μg P4-G1-M1.3, 300 μg P4-G1-M1.3, 100 μgP4-G1-M1.3, anti-IGF-1R+anti-ErbB3 at equal exposure dosing, oranti-IGF-1R+anti-ErbB3 at equimolar dosing (D).

FIG. 58 shows the fitting of the target mediated drug disposition modelto experimental data from mouse blood from mice injected withM1.3-G1-P4. The solid line is the fit of a mouse given a 500 μg dose andthe dotted line is the fit of a mouse given a 100 μg dose.

BRIEF DESCRIPTION OF THE SEQUENCES

The amino acid (“aa”) sequences referred to herein and listed in thesequence listing are identified below.SEQ ID NO:1 is an aa consensus sequence derived from exemplary IGF-1R VHsequences.SEQ ID NO:2 is an aa consensus sequence derived from exemplary IGF-1R VLsequences.SEQ ID NO:3 is an aa consensus sequence derived from exemplary IGF-1R VLsequences, which excludes the VL sequence of the IGF-1R binding site of16F.SEQ ID NO:4 is an aa consensus sequence derived from exemplary ErbB3 VHsequences.SEQ ID NO:5 is an aa consensus sequence derived from exemplary ErbB3 VHsequences, which excludes the VH of the ErbB3 binding site of 16F.SEQ ID NO:6 is an aa consensus sequence derived from exemplary ErbB3 VLsequences.SEQ ID NO:7 is an aa consensus sequence derived from exemplary ErbB3 VLsequences, which excludes the VL sequence of the ErbB3 binding site of16F.SEQ ID NOs:8-31 are the IGF-1R VH aa sequences of FIG. 1.SEQ ID NOs:32-133 are the IGF-1R VL aa sequences of FIG. 2.SEQ ID NOs:134-165 are the ErbB3 VH aa sequences of FIG. 3.SEQ ID NOs:166-200 are the ErbB3 VL aa sequences of FIG. 4.SEQ ID NOs:201-256 are the nucleotide sequence (odd numbers) and aasequences (even numbers) of the mature light and heavy chains ofanti-IGF-1R/anti-ErbB3 IgG1 (scFv)₂ provided in FIG. 5A, the sequence IDnumbers of which are as follows. Kappa light chains: SF (SEQ ID NOs:201and 202); P4 (SEQ ID NOs:203 and 204); M78 (SEQ ID NOs:205 and 206); andM57 (SEQ ID NOs:207 and 208). Heavy chain scFv fusions (hybrids):SF-G1-C8 (i.e., 16F; SEQ ID NOs:209 and 210); SF-G1-P1 (SEQ ID NOs:211and 212); SF-G1-M1.3 (SEQ ID NOs:213 and 214); SF-G1-M27 (SEQ ID NOs:215and 216); SF-G1-P6 (SEQ ID NOs:217 and 218); SF-G1-B69 (SEQ ID NOs:219and 220); P4-G1-C8 (SEQ ID NOs:221 and 222); P4-G1-P1 (SEQ ID NOs:223and 224); P4-G1-M1.3 (SEQ ID NOs:225 and 226); P4-G1-M27 (SEQ ID NOs:227and 228); P4-G1-P6 (SEQ ID NOs:229 and 230); P4-G1-B69 (SEQ ID NOs:231and 232); M78-G1-C8 (SEQ ID NOs:233 and 234); M78-G1-P1 (SEQ ID NOs:235and 236); M78-G1-M1.3 (SEQ ID NOs:237 and 238); M78-G1-M27 (SEQ IDNOs:239 and 240); M78-G1-P6 (SEQ ID NOs:241 and 242); M78-G1-B69 (SEQ IDNOs:243 and 244); M57-G1-C8 (SEQ ID NOs:245 and 246); M57-G1-P1 (SEQ IDNOs:247 and 248); M57-G1-M1.3 (SEQ ID NOs:249 and 250); M57-G1-M27 (SEQID NOs:251 and 252); M57-G1-P6 (SEQ ID NOs:253 and 254) and M57-G1-B69(SEQ ID NOs:255 and 256).SEQ ID NOs:257-296 are the nucleotide sequence (odd numbers) and aasequences (even numbers) of the mature light and heavy chains ofanti-ErbB3/anti-IGF-1R IgG1(scFv)₂ provided in FIG. 5B, the sequence IDnumbers of which are as follows. Lambda light chains: P1 (SEQ ID NOs:257and 258); M27 (SEQ ID NOs:259 and 260); M7 (SEQ ID NOs:261 and 262); B72(SEQ ID NOs:263 and 264); and B60 (SEQ ID NOs:265 and 266). Heavy chainscFv fusions (hybrids): P1-G1-P4 (SEQ ID NOs:267 and 268); P1-G1-M57(SEQ ID NOs:269 and 270); P1-G1-M78 (SEQ ID NOs:271 and 272); M27-G1-P4(SEQ ID NOs:273 and 274); M27-G1-M57 (SEQ ID NOs:275 and 276);M27-G1-M78 (SEQ ID NOs:277 and 278); M7-G1-P4 (SEQ ID NOs:279 and 280);M7-G1-M57 ((SEQ ID NOs:281 and 282); M7-G1-M78 (SEQ ID NOs:283 and 284);B72-G1-P4 (SEQ ID NOs:285 and 286); B72-G1-M57 (SEQ ID NOs:287 and 288);B72-G1-M78 (SEQ ID NOs:289 and 290); B60-G1-P4 (SEQ ID NOs:291 and 292);B60-G1-M57 (SEQ ID NOs:293 and 294); and B60-G1-M78 (SEQ ID NOs:295 and296).SEQ ID NOs:297 and 298 are the nucleotide and aa sequences of the lightchain of 16F with a signal sequence, as shown in FIG. 7B.SEQ ID NOs:299 and 300 are the nucleotide and aa sequences of the heavychain of 16F with a signal sequence, as shown in FIG. 7A.SEQ ID NO:301 is a portion of an exemplary heavy chain domain, wherein alysine was inserted between the C-terminus of the CH3 domain and theN-terminus of the linker SLSLSPGKGGGGS (SEQ ID NO:301—the additionallysine is underlined).SEQ ID NOs:302-304 are consensus sequences of anti-IGF-1R VHCDR1, VHCDR2and VHCDR3 domains, respectively, which are the CDR sequences of the VHconsensus sequence of SEQ ID NO:1 and shown in FIG. 1.SEQ ID NOs:305-307 are consensus sequences of an anti-IGF-1R VLCDR1,VLCDR2 and VLCDR3, respectively, which are the CDR sequences of the VLconsensus sequence of SEQ ID NO:2 and shown in FIG. 2.SEQ ID NO:308 is a consensus sequence of an anti-IGF-1R VLCDR3, which isthe CDR3 sequence of the VL consensus sequence of SEQ ID NO:3 and shownin FIG. 2.SEQ ID NOs:209-311 are consensus sequences of anti-ErbB3 VHCDR1, VHCDR2and VHCDR3 domains, respectively, which are the CDR sequences of the VHconsensus sequence of SEQ ID NO:4 and shown in FIG. 3.SEQ ID NOs:312-314 are consensus sequences of anti-ErbB3 VLCDR1, VLCDR2and VLCDR3 domains, respectively, which are the CDR sequences of the VLconsensus sequence of SEQ ID NO:6 and shown in FIG. 4.SEQ ID NO:315 is a consensus sequence of an anti-ErbB3 VLCDR3, which isthe CDR3 sequence of the VL consensus sequence of SEQ ID NO:7 and shownin FIG. 4.SEQ ID NO:316 is the aa sequence of the heavy chain of theanti-ErbB3/anti-IGF-1R IgG2 tetravalent bispecific protein ELI-7.SEQ ID NO:317 is the aa sequence of the light chain of theanti-ErbB3/anti-IGF-1R IgG2 tetravalent bispecific protein ELI-7.SEQ ID NO:318 is the aa sequence of the heavy chain of theanti-IGF-1R/anti-ErbB3 tetravalent bispecific protein ILE-10.SEQ ID NO:319 is the aa sequence of the heavy chain of theanti-IGF-1R/anti-ErbB3 tetravalent bispecific protein ILE-12.SEQ ID NO:320 is the aa sequence of the light chain of theanti-IGF-1R/anti-ErbB3 tetravalent bispecific proteins ILE-10 andILE-12.SEQ ID NOs:321-335 are the aa sequences of Fab heavy chains (Fab HC),Fab light chains (Fab LC) and scFvs from the anti-IGF-1R antibodies ofTable 1, which sequences are of FIG. 37.

TABLE 1 anti-IGF-1R antibodies Fab Anti-IGF-1R Fab HC Fab LC scFvANTI-IGF-1R SEQ ID NO: 321 SEQ ID NO: 322 SEQ ID NO: 323 Ab#C(figitumumab) ANTI-IGF-1R SEQ ID NO: 324 SEQ ID NO: 325 SEQ ID NO: 326Ab#B (cixutumumab) ANTI-IGF-1R SEQ ID NO: 327 SEQ ID NO: 328 SEQ ID NO:329 Ab# A (ganitumab) BIIB-G11 SEQ ID NO: 330 SEQ ID NO: 331 SEQ ID NO:332 BIIB-C06 SEQ ID NO: 333 SEQ ID NO: 334 SEQ ID NO: 335SEQ ID NOs:336-353 are the aa sequences of Fab heavy chains (Fab HC),Fab light chains (Fab LC) and scFvs from the anti-ErbB3 antibodies ofTable 2, which sequences are of FIG. 38.

TABLE 2 anti-ErbB3 antibodies Fab Anti-ErbB3 Fab HC Fab LC scFvANTI-ErbB3 SEQ ID NO: 336 SEQ ID NO: 337 SEQ ID NO: 338 Ab# A H3 SEQ IDNO: 339 SEQ ID NO: 340 SEQ ID NO: 341 MM Ab#3 SEQ ID NO: 342 SEQ ID NO:343 SEQ ID NO: 344 MM Ab#14 SEQ ID NO: 345 SEQ ID NO: 346 SEQ ID NO: 347MM Ab#17 SEQ ID NO: 348 SEQ ID NO: 349 SEQ ID NO: 350 MM Ab#19 SEQ IDNO: 351 SEQ ID NO: 352 SEQ ID NO: 353SEQ ID NOs:354 and 355 are the nucleotide and aa sequences for theB60-IgG2-M78 polyvalent bispecific antibody shown in FIG. 5B.SEQ ID NOs:356 and 357 are the nucleotide and aa sequences for theM7-IgG2-M78 polyvalent bispecific antibody shown in FIG. 5B.SEQ ID NOs:358-360 are aa sequences of SF, P4, M78, and M57 anti-IGF-1RIgG1 monoclonal antibody heavy chains shown in FIG. 6A.SEQ ID NOs:362-366 are aa sequences of P1, M27, M7, B72, and B60anti-ErbB3 IgG1 monoclonal antibody heavy chains shown in FIG. 6B.SEQ ID NOs:367-369 are aa sequences of P4, M57, and M78 anti-IGF-1R scFvmonoclonal antibodies shown in FIG. 6C.SEQ ID NOs:370-375 are aa sequences of C8, P1, M1.3, M27, P6, and B69anti-ErbB3 scFv monoclonal antibodies shown in FIG. 6D.SEQ ID NOs:376-379 are aa sequences of the heavy chains P4M-G1-M1.3,P4M-G1-C8, P33M-G1-M1.3, and P33M-G1-C8, respectively.SEQ ID NOs:380 and 381 are aa sequences of P33M kappa light chain andP4M kappa light chain, respectively.SEQ ID NOs:382 and 383 are aa sequences of the anti-IGF-1R scFv M76 andthe anti-ErbB3 scFv P6L, respectively. The VH and VL domains of bindingsite M76 consist of the aa sequences of SEQ ID NOs:31 and 133,respectively.SEQ ID NOs:384 and 385 are aa sequences of the VH domain of theanti-IGF-1R binding site modules P4M and P33M, respectively.SEQ ID NOs:386 and 387 are aa sequences of the VL domain of theanti-IGF-1R binding site modules P4M and P33M, respectively.SEQ ID NO:388 is the aa of the VH domain of the anti-ErbB3 binding sitemodule P6L. The VL domain consists of the aa sequence of SEQ ID NO:173.SEQ ID NOs: 389 and 390 are aa sequences of the anti-IGF-1R heavy chainsP4M-G1-P6L and P33M-G1-P6L, respectively.SEQ ID NO:391 is the aa sequence of the anti-ErbB3 heavy chainP1-G1-M76.SEQ ID NO:392 is the aa sequence of the beginning of the CH1 portion ofthe hybrid heavy chains of FIGS. 5A and 5B.SEQ ID NO:393 and 394 are aa sequences of the beginning of the CL domainin the anti-IGF-1R and ErbB3 VL domains of the light chains of FIGS. 5Aand 5B, respectively.SEQ ID NOs:395-402 are the aa sequence of exemplary Gly-Ser polypeptidelinkers.SEQ ID NO:403 is the aa sequence of a hexa-histidine tag.SEQ ID NO:404 is the aa sequence of the IgG2 constant domain (includingCH1, Hinge, CH2, and CH3 regions).SEQ ID NOs:405-408 are the aa sequence of the heavy chains of SF-G1-P1,SF-G1-M27, M57-G1-C8, M7-G1-M78 heavy chains, respectively, includingthe leader sequence (the N-terminal 19 aas of each sequence).SEQ ID NO:409 is the nucleotide sequence of the M7-G1-M78 heavy chain,including the leader sequence. SEQ ID NOs:410-411 are the aa andnucleotide sequences of the P4-G1-M1.3 heavy chain, respectively,including the leader sequence (the N-terminal 19 aas of the aasequence).SEQ ID NOs:412-413 are the aa and nucleotide sequences of the P4-G1-C8heavy chain, respectively, including the leader sequence (the N-terminal19 aas of the aa sequence).SEQ ID NOs:414-415 are the aa and nucleotide sequences of the M7 LambdaLight Chain, respectively, including the leader sequence (the N-terminal21 aas of the aa sequence).SEQ ID NOs:416-417 are the aa and nucleotide sequences of the P4 KappaLight Chain, respectively, including the leader sequence (the N-terminal20 aas of the aa sequence).SEQ ID NO:418 is the aa sequence of the IgG1 module with Hinge, CH2, andCH3 regions (the C-terminal 231 aas of the sequence).SEQ ID NOs:419-424 are nucleotide sequences of the heavy chainsP4M-G1-M1.3, P4M-G1-C8, P4M-G1-P6L, P33M-G1-M1.3, P33M-G1-C8, andP33M-G1-P6L respectively.SEQ ID NO:425 is the nucleotide sequence of the P1-G1-M76anti-ErbB3-G1/anti-EGF-1R bispecific antibody.SEQ ID NOs:426 and 427 are nucleotide sequences of the P33M Kappa andP4M Kappa light chains, respectively.SEQ ID NO:428 is the nucleotide sequence of the M76 anti-IGF-1R scFv.SEQ ID NO:429 is the nucleotide sequence of the P6L anti-ErbB3 scFv.

DETAILED DESCRIPTION

Provided herein are novel monospecifc antibodies that bind specificallyto IGF-1R or to ErbB3. Such antibodies include IgG antibodies and scFvantibodies. Further provided are bispecific antibodies, e.g., polyvalentbispecific antibodies (“PBAs”) that bind specifically to human IGF-1Rand to human ErbB3. These proteins are potent inhibitors of tumor cellproliferation and of signal transduction through either or both ofIGF-1R and ErbB3. The proteins may be used for treating a cellproliferative disorder, e.g., a cancer.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below.

“Agent,” refers to an active molecule, e.g., a therapeutic protein,e.g., a drug.

“Aa substitution” refers to the replacement of one specific aa (“aa”) ina protein with another aa. A substitution may be a conservativesubstitution, as defined below.

“Anti-ErbB3 binding site” refers to a binding site that bindsspecifically to human ErbB3.

“Anti-IGF-1R binding site” refers to a binding site that bindsspecifically to human IGF-1R.

“Antigen binding site” refers to a binding site that comprises the VHand/or VL domain of an antibody, or at least one CDR thereof. Forexample, an antigen binding site may comprise, consist essentially of,or consist of a VHCDR3 alone or together with a VHCDR2 and optionally aVHCDR1. In certain embodiments, an antigen binding site comprises a VHdomain and a VL domain, which may be present on the same polypeptide oron two different polypeptides, e.g., the VH domain is present on a heavychain and a VL domain is present on a light chain.

“Antigen-binding portion” of an antibody refers to one or more fragmentsof an antibody that retain the ability to specifically bind to anantigen (e.g., IGF-1R or ErbB3). It has been shown that theantigen-binding function of an antibody can be retained by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) an Fdfragment consisting of the VH and CH1 domains; (iv) an Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although VL andVH are two domains of an Fv fragment, VL and VH are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent proteins, knownas single chain Fvs (scFvs) see U.S. Pat. No. 5,892,019. Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites.

“Binding affinity” refers to the strength of a binding interaction andincludes both the actual binding affinity as well as the apparentbinding affinity. The actual binding affinity is a ratio of theassociation rate over the disassociation rate. The apparent affinity caninclude, for example, the avidity resulting from a polyvalentinteraction. Dissociation constant (K_(d)), is typically the reciprocalof the binding affinity, and may be conveniently measured using asurface plasmon resonance assay (e.g., as determined in a BIACORE 3000instrument (GE Healthcare) e.g., using recombinant ErbB3 as the analyteand an anti-ErbB3 antibody as the ligand) or a cell binding assay, eachof which assays is described in Example 3 of U.S. Pat. No. 7,846,440.

“Binding moiety,” “binding domain,” or “binding site,” refers to theportion, region, or site of a binding polypeptide or, when so specified,of a heavy or light chain thereof, that is directly involved inmediating the specific binding of an antibody to a target molecule (i.e.an antigen). Exemplary binding domains include an antigen binding site,a receptor binding domain of a ligand, a ligand binding domain of areceptor or an enzymatic domain. In preferred embodiments, the bindingdomain comprises or consists of an antigen binding site (e.g.,comprising a variable heavy (VH) chain sequence and variable light (VL)chain sequence or six CDRs from an antibody placed into alternativeframework regions (e.g., human framework regions optionally comprisingone or more aa substitutions). In certain embodiments, a binding sitemay be comprised essentially only of a VH or a VL chain sequence. Abinding site may be entirely from one species, e.g., it has onlysequences that derive from the germline sequences of one species. Forexample, a binding site may be human (i.e., from the human species),mouse, or rat. A binding site may also be humanized, i.e., the CDRs arefrom one species and the frameworks (FRs) are from another species. Forexample, a binding site may have CDRs that were derived from a mouseantibody and FRs that are from the human species. Certain humanizedbinding sites comprise mutations in one or more CDR to make the CDRslook more like the CDRs of the donor antibody. Certain humanizedantibodies may also comprise mutations in one or more FR. Generallymutations in a binding site may enhance the affinity of binding of thebinding site to its target antigen, and/or they may stabilize thebinding site, e.g., to extend its half-life.

“CDR” or “complementarity determining region” refers to thenoncontiguous antigen combining sites found within the VR of both heavyand light chain polypeptides. These particular regions have beendescribed by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) andKabat et al., Sequences of protein of immunological interest. (1991),and by Chothia et al., J. Mol. Biol. 196:901-917 (1987) and by MacCallumet al., J. Mol. Biol. 262:732-745 (1996) where the definitions includeoverlapping or subsets of aa residues when compared against each other.The aa residues which encompass the CDRs as defined by each of the abovecited references are set forth for comparison. As used herein, and ifnot otherwise specified, “CDR” is as defined by Kabat.

TABLE 3 CDR definitions CDR Definitions Kabat¹ Chothia² MacCallum³VHCDR1 31-35 26-32 30-35 VHCDR2 50-65 53-55 47-58 VHCDR3  95-102  96-101 93-101 VLCDR1 24-34 26-32 30-36 VLCDR2 50-56 50-52 46-55 VLCDR3 89-9791-96 89-96 ¹Residue numbering follows the nomenclature of Kabat et al.,1991, supra ²Residue numbering follows the nomenclature of Chothia etal., supra ³Residue numbering follows the nomenclature of MacCallum etal., supra

“CH1 domain” refers to the heavy chain immunoglobulin constant domainlocated between the VH domain and the hinge. It spans EU positions118-215. A CH1 domain may be a naturally occurring CH1 domain, or anaturally occurring CH1 domain in which one or more aas have beensubstituted, added or deleted, provided that the CH1 domain has thedesired biological properties. A desired biological activity may be anatural biological activity, an enhanced biological activity or areduced biological activity relative to the naturally occurringsequence.

“CH2 domain” refers to the heavy chain immunoglobulin constant domainthat is located between the hinge and the CH3 domain. It spans EUpositions 231-340. A CH2 domain may be a naturally occurring CH2 domain,or a naturally occurring CH2 domain in which one or more aas have beensubstituted, added or deleted, provided that the CH2 domain has thedesired biological properties. A desired biological activity may be anatural biological activity, an enhanced biological activity or areduced biological activity relative to the naturally occurringsequence.

“CH3 domain” refers to the heavy chain immunoglobulin constant domainthat is located C-terminally of the CH2 domain and spans approximately110 residues from the N-terminus of the CH2 domain, e.g., aboutpositions 341-446b (EU numbering system). A CH3 domain may be anaturally occurring CH3 domain, or a naturally occurring CH3 domain inwhich one or more aas (“aas”) have been substituted, added or deleted,provided that the CH3 domain has the desired biological properties. Adesired biological activity may be a natural biological activity, anenhanced biological activity or a reduced biological activity relativeto the naturally occurring sequence. A CH3 domain may or may notcomprise a C-terminal lysine.

“CH4 domain” refers to the heavy chain immunoglobulin constant domainthat is located C-terminally of the CH3 domain in IgM and IgEantibodies. A CH4 domain may be a naturally occurring CH4 domain, or anaturally occurring CH4 domain in which one or more aas have beensubstituted, added or deleted, provided that the CH4 domain has thedesired biological properties. A desired biological activity may be anatural biological activity, an enhanced biological activity or areduced biological activity relative to the naturally occurringsequence.

“CL domain” refers to the light chain immunoglobulin constant domainthat is located C-terminally to the VH domain. It spans about Kabatpositions 107A-216. A CL domain may be a naturally occurring CL domain,or a naturally occurring CL domain in which one or more aas have beensubstituted, added or deleted, provided that the CL domain has thedesired biological properties. A desired biological activity may be anatural biological activity, an enhanced biological activity or areduced biological activity relative to the naturally occurringsequence. A CL domain may or may not comprise a C-terminal lysine.

“Conservative substitution” or “conservative aa substitution” refers tothe replacement of one or more aa residues in a protein or a peptidewith, for each particular pre-substitution aa residue, a specificreplacement aa that is known to be unlikely to alter either theconfirmation or the function of a protein or peptide in which such aparticular aa residue is substituted for by such a specific replacementaa. Such conservative substitutions typically involve replacing one aawith another that is similar in charge and/or size to the first aa, andinclude replacing any of isoleucine (I), valine (V), or leucine (L) foreach other, substituting aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions are known inthe art to be conservative in particular sequence or structuralenvironments. For example, glycine (G) and alanine (A) can frequently besubstituted for each other to yield a conservative substitution, as canbe alanine and valine (V). Methionine (M), which is relativelyhydrophobic, can frequently conservatively substitute for or beconservatively substituted by leucine or isoleucine, and sometimesvaline Lysine (K) and arginine (R) are frequently interchangeable inlocations in which the significant feature of the aa residue is itscharge and the differing pK's of these two basic aa residues are notexpected to be significant. The effects of such substitutions can becalculated using substitution score matrices such PAM120, PAM-200, andPAM-250. Other such conservative substitutions, for example,substitutions of entire regions having similar hydrophobicitycharacteristics (e.g., transmembrane domains), are well known.

A CR domain on a light chain of an immunoglobulin is referred tointerchangeably as a “CL,” “light chain CR domain,” “CL region” or “CLdomain.” A constant domain on a heavy chain (e.g., hinge, CH1, CH2 orCH3 domains) of an immunoglobulin is referred to interchangeably as a“CH,” “heavy chain constant domain,” “CH” region or “CH domain.” Avariable domain on an immunoglobulin light chain is referred tointerchangeably as a “VL,” “light chain variable domain,” “VL region” or“VL domain.” A variable domain on an immunoglobulin heavy chain isreferred to interchangeably as a “VH,” “heavy chain variable domain,”“VH region” or “VH domain.”

“Domain” refers to a region, e.g., an independently folding, globularregion or a non-globular region (e.g., a linker domain), of a heavy orlight chain polypeptide which may comprise peptide loops (e.g., 1 to 4peptide loops) that may be stabilized, for example, by a β-pleated sheetand/or an intrachain disulfide bond. The constant and VRs ofimmunoglobulin heavy and light chains are typically folded into domains.In particular, each one of the CH1, CH2, CH3, CH4, CL, VH and VL domainstypically form a loop structure.

“EC₅₀” or “EC50” refers to the concentration of a molecule, e.g., a PBA,that provides 50% of the maximal effect of the protein on a particularsystem such as a binding assay or a signal transduction pathway.

“ErbB3” and “HER3” refer to ErbB3 protein, as described in U.S. Pat. No.5,480,968. The human ErbB3 protein sequence is shown in FIG. 4 and SEQID NO:4 of U.S. Pat. No. 5,480,968, wherein the first 19 aas correspondto the leader sequence that is cleaved from the mature protein. ErbB3 isa member of the ErbB family of receptors, other members of which includeErbB1 (EGFR), ErbB2 (HER2/Neu) and ErbB4. While ErbB3 itself lackstyrosine kinase activity, but is itself phosphorylated upon dimerizationof ErbB3 with another ErbB family receptor, e.g., ErbB1, ErbB2 andErbB4, which are receptor tyrosine kinases. Ligands for the ErbB familyinclude heregulin (HRG), betacellulin (BTC), epidermal growth factor(EGF), heparin-binding epidermal growth factor (HB-EGF), transforminggrowth factor alpha (TGF-α), amphiregulin (AR), epigen (EPG) andepiregulin (EPR). The aa sequence of human ErbB3 is provided at GenbankAccession No. NP_(—)001973.2 (receptor tyrosine-protein kinase erbB-3isoform 1 precursor) and is assigned Gene ID: 2065.

“EU” indicates that aa positions in a heavy chain CR, including aapositions in the CH1, hinge, CH2, and CH3 domains, are numbered hereinaccording to the EU index numbering system (see Kabat et al., in“Sequences of Proteins of Immunological Interest”, U.S. Dept. Health andHuman Services, 5^(th) edition, 1991).

“Fab” refers to the antigen binding portion of an antibody, comprisingtwo chains: a first chain that comprises a VH domain and a CH1 domainand a second chain that comprises a VL domain and a CL domain. Althougha Fab is typically described as the N-terminal fragment of an antibodythat was treated with papain and comprises a portion of the hingeregion, it is also used herein as referring to a binding domain whereinthe heavy chain does not comprise a portion of the hinge.

“Fc region” refers to the portion of a single immunoglobulin heavy chainbeginning in the hinge region just upstream of the papain cleavage site(i.e. residue 216 in IgG, taking the first residue of heavy chain CR tobe 114) and ending at the C-terminus of the antibody. Accordingly, acomplete Fc region comprises at least a hinge, a CH2 domain, and a CH3domain. Two Fc regions that are dimerized are referred to as “Fc” or “Fcdimer.” An Fc region may be a naturally occurring Fc region, or anaturally occurring Fc region in which one or more aas have beensubstituted, added or deleted, provided that the Fc region has thedesired biological properties. A desired biological activity may be anatural biological activity, an enhanced biological activity or areduced biological activity relative to the naturally occurringsequence.

“Framework region” or “FR” or “FR region” includes the aa residues thatare part of the VR, but are not part of the CDRs (e.g., using the Kabatdefinition of CDRs). Therefore, a VR framework is between about 100-120aas in length but includes only those aas outside of the CDRs. For thespecific example of a heavy chain VR and for the CDRs as defined byKabat et al., 1991, ibid., framework region 1 corresponds to the domainof the VR encompassing aas 1-30; framework region 2 corresponds to thedomain of the VR encompassing aas 36-49; framework region 3 correspondsto the domain of the VR encompassing aas 66-94, and framework region 4corresponds to the domain of the VR from aas 103 to the end of the VR.The framework regions for the light chain are similarly separated byeach of the light chain VR CDRs. Similarly, using the definition of CDRsby Chothia et al. or McCallum et al. the framework region boundaries areseparated by the respective CDR termini as described above. In preferredembodiments, the CDRs are as defined by Kabat.

“Full-length antibody” is an antibody that comprises one or more heavychains and one or more light chains. Each heavy chain is comprised of aheavy chain VR (abbreviated herein as VH) and a heavy chain CR. Theheavy chain CR is comprised of three domains CH1, CH2, and CH3, andoptionally a fourth domain, CH4. Each light chain is comprised of alight chain VR (abbreviated herein as VL) and a light chain CR. Thelight chain CR is comprised of one domain, CL. The VH and VL regions canbe further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis typically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulin proteins can be of anytype class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or, subclass (e.g.,IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.

“Gly-Ser linker” or “Gly-Ser peptide” refers to a peptide that consistsof glycine and serine residues. An exemplary Gly-Ser peptide comprisesthe aa sequence (Gly₄Ser)n (SEQ ID NO:395), wherein n=1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In certainembodiments, n is a number between 1 and 5, n is a number between 6 and10, n is a number between 11 and 15, n is a number between 16 and 20, nis a number between 21 and 25, or n is a number between 26 and 30.

“Heavy chain immunoglobulin CR” or “HC Ig CR” may comprise a CH1 domainand an Fc region, which Fc region may comprise a hinge, a CH2 domain, aCH3 domain and/or a CH4 domain. A light chain immunoglobulin CR maycomprise a CL domain.

“Hinge” or “hinge region” or “hinge domain” refers to the flexibleportion of a heavy chain located between the CH1 domain and the CH2domain. It is approximately 25 aas long, and is divided into an “upperhinge,” a “middle hinge,” and a “lower hinge.” A hinge may be anaturally occurring hinge, or a naturally occurring hinge in which oneor more aas have been substituted, added or deleted, provided that thehinge has the desired biological properties. A desired biologicalactivity may be a natural biological activity, an enhanced biologicalactivity or a reduced biological activity relative to the naturallyoccurring sequence.

“IC₅₀,” or “IC50” refers to the concentration of a molecule, e.g., aPBA, that provides a 50% inhibition of a maximal activity (e.g., aresponse to a stimulus or a constitutive activity), i.e., aconcentration that reduces the activity to a level halfway between themaximal activity and the baseline. The IC₅₀ value may be converted to anabsolute inhibition constant (Ki) using, e.g., the Cheng-Prusoffequation. In a system that is inhibited by a binding agent, such as anantibody or a bispecific binding protein provided herein, the IC50 maybe indistinguishable from the EC50.

“IGF-1R” or “IGF1R” refers to the receptor for insulin-like growthfactor 1 (IGF-1, formerly known as somatomedin C). IGF-1R also binds to,and is activated by, insulin-like growth factor 2 (IGF-2). IGF1-R is areceptor tyrosine kinase, which upon activation by IGF-1 or IGF-2 isauto-phosphorylated. The aa sequence of human IGF-1R precursor isprovided at Genbank Accession No. NP_(—)000866 and is assigned Gene ID:3480.

“IgG-(scFv)₂” indicates a tetravalent PBA consisting of an IgG havingtwo N-terminal Fab binding sites each comprised of an IgG heavy chainand an IgG light chain, wherein the C-terminus of each heavy chain islinked to an scFv having a binding site comprised of a VH domain and aVL domain. When the immunoglobulin CRs are those of an IgG1, the PBA isreferred to as an “IgG1-(scFv)₂.” Exemplary IgG1-(scFv)₂ PBAs are thosewhere the four binding sites comprise two essentially identicalanti-IGF-1R binding sites and two essentially identical anti-ErbB3binding sites. The 38 tetravalent PBAs set forth below in the DetailedDescription under the subheading “Exemplary IGF-1R+ErbB3 PBAs comprisingIgG1 CRs” (also see FIGS. 5A and B), each comprise two joinedessentially identical subunits, each subunit comprising a heavy and alight chain that are disulfide bonded to each other, e.g., M7-G1-M78(SEQ ID NO:284 and SEQ ID NO:262), P4-G1-M1.3 (SEQ ID NO:226 and SEQ IDNO:204), and P4-G1-C8(SEQ ID NO:222 and SEQ ID NO:204), are exemplaryembodiments of such IgG1-(scFv)₂ proteins. When the immunoglobulin CRsare those of IgG2, the protein is referred to as an “IgG2-(scFv)₂.” Anexemplary “IgG2-(scFv)₂ protein is ELI-7. When the immunoglobulin CRsare partially from an IgG1 and partially from another isotype of IgG,e.g., an IgG2, the protein is referred to as e.g., an “IgG1/2-(scFv)₂.”

“Immunoglobulin CR” or “Ig CR” refers to the parts of an immunoglobulin,(i.e., an antibody,) outside of its variable domains. In certainembodiments, an immunoglobulin CR comprises a “heavy chainimmunoglobulin CR” and a “light chain immunoglobulin CR.”

“Inhibition” of a biological activity by a binding protein refers to anyreproducibly detectable decrease in biological activity mediated by thebinding protein. In some embodiments, inhibition provides astatistically significant decrease in biological activity, e.g., adecrease of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% in biological activity relative to the biological activitydetermined in he absence of the binding protein.

“Isolated,” in reference to polynucleotides, polypeptides or proteins,means that the polynucleotide, polypeptide or protein is substantiallyremoved from polynucleotides, polypeptides, proteins or othermacromolecules with which it, or its analogues, occurs in nature.Although the term “isolated” is not intended to require a specificdegree of purity, typically, the protein will be at least about 75%pure, more preferably at least about 80% pure, more preferably at leastabout 85% pure, more preferably at least about 90% pure, more preferablystill at least about 95% pure, and most preferably at least about 99%pure.

“Kabat” in conjunction with designation of immunoglobulin aa sequencepositions indicates that aa positions in a light chain CR (e.g., CLdomain) are numbered according to the Kabat index numbering system (seeKabat et al., 1991, op. cit.).

“Linked to” refers to direct or indirect linkage or connection of, incontext, aas or nucleotides. An “indirect linkage” refers to a linkagethat is mediated through a linker or a domain, comprising, e.g., one ormore aas or nucleotides. A “direct linkage” or “linked directly” whenreferring to two polypeptide segments refers to the presence of covalentbond between the two polypeptide segments, e.g., the two polypeptidesegments are joined contiguously without intervening sequences.

“Linker” refers to one or more aas connecting two domains or regionstogether. A linker may be flexible to allow the domains being connectedby the linker to form a proper three dimensional structure therebyallowing them to have the required biological activity. A linkerconnecting the VH and the VL of an scFv is referred to herein as an“scFv linker” A linker connecting the N-terminus of a VH domain or theC-terminus of the CH3 domain to a second VH domain, e.g., that of anscFv is referred to as a “connecting linker”

“Module” refers to a structurally and/or functionally distinct part of aPBA, such a binding site (e.g., an scFv domain or a Fab domain) and theIg constant domain. Modules provided herein can be rearranged (byrecombining sequences encoding them, either by recombining nucleic acidsor by complete or fractional de novo synthesis of new polynucleotides)in numerous combinations with other modules to produce a wide variety ofPBAs, e.g., as disclosed herein. For example, an “SF” module refers tothe binding site “SF,” i.e., comprising at least the CDRs of the SF VHand SF VL domains. A “C8” module refers to the binding site “C8.”

“PBA” refers to a polyvalent bispecific antibody, an artificial hybridprotein comprising at least two different binding moieties or domainsand thus at least two different binding sites (e.g., two differentantibody binding sites), wherein one or more of the pluralities of thebinding sites are covalently linked, e.g., via peptide bonds, to eachother. A preferred PBA described herein is an anti-IGF-1R+anti-ErbB3PBA, which is a polyvalent bispecific antibody that comprises one ormore first binding sites binding specifically to an IGF-1R protein,e.g., a human IGF-1R protein, and one or more second binding sitesbinding specifically to an ErbB3 protein, e.g., a human ErbB3 protein.An anti-IGF-1R+anti-ErbB3 PBA is so named regardless of the relativeorientations of the anti-IGF-1R and anti-ErbB3 binding sites in themolecule, whereas when the PBA name comprises two antigens separated bya slash (/) the antigen to the left of the slash is amino terminal tothe antigen tot the right of the slash. A PBA may be a bivalent bindingprotein, a trivalent binding protein, a tetravalent binding protein or abinding protein with more than 4 binding sites. An exemplary PBA is atetravalent bispecific antibody, i.e., an antibody that has 4 bindingsites, but binds to only two different antigens or epitopes. Exemplarybispecific antibodies are tetravalent “anti-IGF-1R/anti-ErbB3” PBAs and“anti-ErbB3/anti-IGF-1R” PBAs. Typically the N-terminal binding sites ofa tetravalent PBA are Fabs and the C-terminal binding sites are scFvs.

“Percent identical” or “% identical” refers to two or more nucleic acidor polypeptide sequences or subsequences that are the same (100%identical) or have a specified percentage of nucleotide or aa residuesthat are the same, when the two sequences are aligned for maximumcorrespondence and compared. To align for maximum correspondence, gapsmay be introduced into one of the sequences being compared. The aaresidues or nucleotides at corresponding positions are then compared andquantified. When a position in the first sequence is occupied by thesame residue as the corresponding position in the second sequence, thenthe sequences are identical at that position. The percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (e.g., % identity=# of identicalpositions/total # of positions (e.g., overlapping positions)×100). Incertain embodiments, the two sequences are the same length. Thedetermination that one sequence is a measured % identical with anothersequence can be determined using a mathematical algorithm. Anon-limiting example of a mathematical algorithm utilized for suchcomparison of two sequences is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program e.g., for comparing aasequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 may be used. Additional algorithms for sequenceanalysis are well known in the art and many are available online.

“Portion” or “fragment” (e.g., of a domain) of a reference moiety refersto a discrete part of the whole reference moiety (e.g., domain, e.g., anaturally occurring domain) that is at least, or at most 10% 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the size of thereference moiety.

“scFv linker” refers to a peptide or polypeptide domain interposedbetween the VL and VH domains of an scFv. scFv linkers preferably alloworientation of the VL and VH domains in a antigen binding conformation.In one embodiment, an scFv linker comprises or consists of a peptide orpolypeptide linker that only comprises glycines and serines (a “Gly-Serlinker”). In certain embodiments, an scFv linker comprises a disulfidebond.

“scFv protein” refers to a binding protein that consists of a singlepolypeptide comprising one light chain variable domain (VL), and oneheavy chain variable domain (VH), wherein each variable domain isderived from the same or different antibodies. scFv proteins typicallycomprise an scFv linker interposed between the VH domain and the VLdomain. ScFv proteins are known in the art and are described, e.g., inU.S. Pat. No. 5,892,019.

“Similarity” or “percent similarity” in the context of two or morepolypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of aa residues that are the same orconservatively substituted when compared and aligned for maximumcorrespondence. By way of example, a first aa sequence can be consideredsimilar to a second aa sequence when the first aa sequence is at least50%, 60%, 70%, 75%, 80%, 90%, or even 95% identical, or conservativelysubstituted, to the second aa sequence when compared to an equal numberof aas as the number contained in the first sequence, or when comparedto an alignment of polypeptides that has been aligned by a computersimilarity program known in the art. These terms are also applicable totwo or more polynucleotide sequences.

“Specific binding,” “specifically binds,” “selective binding,” and“selectively binds,” as well as “binds specifically” “bindsselectively,” when referring to the binding of a binding site to itstarget epitope or a combination of binding sites to their targetepitopes, means that the binding site(s) exhibit(s) immunospecificbinding to the target epitope(s). A binding site that binds specificallyto an epitope exhibits appreciable affinity for a target epitope and,generally, does not exhibit cross-reactivity with other epitopes in thatit does not exhibit appreciable affinity to any unrelated epitope andpreferably does not exhibit affinity for any unrelated epitope that isequal to, greater than, or within two orders of magnitude lower than theaffinity for the target epitope. “Appreciable” or preferred bindingincludes binding with a dissociation constant (Kd) of 10⁻⁸, 10⁻⁹ M,10⁻¹⁰, 10⁻¹¹, 10⁻¹² M, 10⁻¹³ M or an even lower Kd value. Note thatlower values for Kd (dissociation constant) indicate higher bindingaffinity, thus a Kd of 10⁻⁷ is a higher Kd value than a Kd of 10⁻⁸, butindicates a lower binding affinity than a Kd of 10⁻⁸). Dissociationconstants with values of about 10⁻⁷ M, and even as low as about 10⁻⁸ M,are at the high end of dissociation constants suitable for therapeuticantibodies. Binding affinities may be indicated by a range ofdissociation constants, for example, 10⁻⁶ to 10⁻¹² M, 10⁻⁷ to 10⁻¹² M,10⁻⁸ to 10⁻¹² M or better (i.e., or lower value dissociation constant).Dissociation constants in the nanomolar (10⁻⁹ M) to picomolar (10⁻¹² M)range or lower are typically most useful for therapeutic antibodies.Suitable dissociation constants are Kds of 50 nM or less (i.e., abinding affinity of 50 nM or higher—e.g., a Kd of 45 nM) or Kds of 40nM, 30 nM, 20 nM, 10 nM, 1 nm, 100 pM, 10 pM or 1 pM or less. Specificor selective binding can be determined according to any art-recognizedmeans for determining such binding, including, for example, according toScatchard analysis and/or competitive binding assays.

Polyvalent Bispecific Antibodies

Provided herein are polyvalent bispecific antibodies (“PBAs”), which maybe isolated monoclonal antibodies. Exemplary PBAs comprise at least oneanti-IGF-1R binding site and at least one anti-ErbB3 binding site or atleast two anti-IGF-1R binding sites and at least two anti-ErbB3 bindingsites. In a preferred embodiment, the anti-IGF-1R binding site bindsspecifically to a human IGF-1R and the anti-ErbB3 binding site bindsspecifically to human ErbB3. In certain embodiments, the PBA comprisestwo heavy-light chain pairs that associate with each other to form asingle protein, wherein each heavy-light chain pair comprises ananti-IGF-1R binding site and an anti-ErB3 binding site. In certainembodiments, the anti-IGF-1R binding site and the anti-ErbB3 bindingsite of a first heavy-light chain pair are connected through animmunoglobulin CR that associates with the immunoglobulin CR of anotherheavy-light chain pair (e.g., by disulfide bonds) to form, e.g., asingle IgG-like protein. A preferred PBA as described herein hasadvantageous properties, such as the ability to inhibit tumor cellproliferation and to reduce or stabilize tumor growth equivalently to ormore potently than either its isolated anti-IGF-1R binding moiety or itsisolated anti-ErbB3 binding moiety, and in certain embodiments, theability to inhibit either or both of tumor invasiveness and tumormetastasis. An exemplary PBA described herein can inhibit either or bothof IGF-1R and ErbB3 mediated signal transduction, such as IGF-1R, ErbB3and AKT phosphorylation, equivalently to or more potently than eitherits isolated anti-IGF-1R binding moiety or its isolated anti-ErbB3binding moiety. An exemplary PBA will (i) inhibit growth of tumor cells,e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more;or (ii) inhibit IGF-1r, ErbB3 or Akt phosphorylation, e.g., by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, e.g., to a similarextent or more potently than either its isolated anti-IGF-1R bindingmoiety or its isolated anti-ErbB3 binding moiety, or both (i) and (ii).An exemplary PBA will (iii) be stable, e.g., be at least 80% monomericin a solution after 1, 2, 3, 4, 5 or more days at 4° C., roomtemperature or 37° C., or (iv) have a Tm (e.g., as determined by DSF) ofat least 50° C., 55° C., 60° C., 65° C. or more, or both (iii) and (iv).The PBAs described herein may be used, e.g., for treating a subjecthaving a cancer.

In certain embodiments, the immunoglobulin CR of a PBA may comprise theCR of an IgG heavy chain, which may comprise an Fc region. Animmunoglobulin constant domain may exist as a heavy-light chain pair,the heavy chain Fc region of which may comprise a CH3 domain thatassociates (e.g., by disulfide bonds) with the CH3 domain of anothersuch heavy-light chain pair. The immunoglobulin CR moiety may alsocomprise a CH2 domain, a hinge and/or a CH1 domain. As further describedherein, each of a CH1, hinge, CH2, or CH3 domain of a PBA may be anaturally occurring (or wild type) domain, or it may differ from anaturally occurring domain by one or more aa substitutions (e.g.,conservative substitutions), additions or deletions, provided that theparticular domain retains its desired biological activity, such aseither or both of CH3 and CL association activity. When present, theCH1, hinge, CH2 and CH3 domains are preferably in N- to C-terminal orderas they occur naturally, i.e., CH1, hinge, CH2, CH3. These domains maybe connected, or linked, to each other directly or indirectly. Anindirect linkage is a linkage that is mediated through a linker of oneor more aas. In one embodiment, each CH domain is directly linked to itsadjacent domains. Accordingly, in one embodiment, a CH1 domain is linkedat its C-terminus to the N-terminus of a hinge domain, which is linkedat its C-terminus to the N-terminus of a CH2 domain, which is linked atits C-terminus to the N-terminus of a CH3 domain.

Certain PBAs comprise at least two anti-IGF-1R and at least twoanti-ErbB3 binding sites, each of which bind specifically to IGF-1R orErbB3, respectively. The binding sites may be any type ofimmunoglobulin-derived or mimetic binding site, provided that eachbinding site binds specifically to its respective target. For example, abinding site may be a Fab domain, an scFv, or a fragment of a singledomain antibody. The anti-IGF-1R and anti-ErbB3 binding sites of a PBAmay be the same type of binding site or a different type. For example,the anti-IGF-1R binding sites may be Fabs and the anti-ErbB3 bindingsites may be scFvs. Alternatively, the anti-IGF-1R binding sites may bescFvs and the anti-ErbB3 binding sites may be Fabs. In anotherembodiment, one anti-ErbB3 binding site is a Fab and another anti-ErbB3binding site is an scFv; in another embodiment, one anti-IGF-1R bindingsite is a Fab and another anti-IGF-1R binding site is an scFv. In someembodiments, a first and a second Fab are linked to the N-terminus andC-terminus of the immunoglobulin CR domain, respectively. In someembodiments, a first and a second scFv are linked to the N-terminus andC-terminus of the immunoglobulin CR domain, respectively. In someembodiments, at least one Fab domain is linked to the N-terminus of theimmunoglobulin CR (e.g., in the Fab and CRs' natural arrangement) and atleast one scFv is linked to the C-terminus of the immunoglobulin CR. Insome embodiments, at least one scFv is linked to the N-terminus of theimmunoglobulin CR and at least one Fab is linked to the C-terminus ofthe immunoglobulin CR. Exemplary arrangements of Fab and scFv andanti-IGF-1R and anti-ErbB3 bispecific antibodies are of Table 4.

TABLE 4 Exemplary anti-IGF-1R and anti-ErbB3 arrangements in PBAsLinkage to the N-terminus of the Immunoglobulin CR anti-IGF-1RAnti-IGF-1R Anti-ErbB3 Anti-ErbB3 scFv Fab scFv Fab Linkage toAnti-IGF-1R yes yes the C- scFv terminus of Anti-IGF-1R yes yes theImmuno- Fab globulin CR Anti-ErbB3 yes yes scFv Anti-ErbB3 yes yes Fab

In certain embodiments, the immunoglobulin CR of a PBA comprises a CH1domain that is linked to a first heavy chain variable domain (VH)domain. For example the CH1 domain may be linked at the N-terminus tothe C-terminus of a first VH domain.

In certain embodiments, the immunoglobulin CR of a PBA comprises a CH3that is linked to a second VH domain. When referring to first and secondbinding sites of an IgG based (e.g., derived from or comprising at leastpart of the CR of and IgG) PBA provided herein, the “first” binding siterefers to the binding site that is located N-terminally to theimmunoglobulin CR moeity, whereas the “second” binding site is thebinding site that is located C-terminally to the immunoglobulin CRmoeity. For example a CH3 domain can be linked at its C-terminus to theN-terminus of a second VH domain. The CH3 domain may be linked at its Cterminus to the N-terminus of a linker, which linker is linked at itsC-terminus to the N-terminus of the second VH domain. Such a linker maybe useful to provide flexibility between the constant immunoglobulinregion and the second VH domain, such that a proper three-dimensionalstructure may be obtained to allow the protein to have a biologicalactivity.

In certain embodiments, a PBA comprises two binding sites that areantigen-binding sites as typically found in antibodies (i.e., itcomprises two Fabs). Such PBAs usually comprise two light chains,wherein each light chain comprises a light chain variable (VL) domainthat associates with (e.g., by disulfide binding) the VH domain of eachof two heavy chains, to form two binding sites. The VL domain may belinked to a constant light chain (CL) domain and form a light chain Fabregion. For example, a VL domain may be linked at its C-terminus to theN-terminus of a CL domain. In embodiments in which the first and thesecond binding sites are Fabs, the PBA has two different light chains,referred to as a first and a second light chain, wherein the first andthe second light chains comprise a first and a second VL domain,respectively, and optionally a first and a second CL domain,respectively, and associate (e.g., dimerize) with the first and thesecond VH domain and optionally a first and a second CH1 domain,respectively.

In embodiments in which a PBA comprises one or more scFvs, the VH domainof each scFv is linked to an scFv linker, which is linked to a VLdomain, and a VH domain and a VL domain associate with each other toform an antigen binding site. In one embodiment, a VH domain is linkedat its C-terminus to the N-terminus of an scFv linker, which is linkedat its C-terminus to the N-terminus of a VL domain. In embodiments inwhich one scFv is linked to the N-terminus of an immunoglobulin CR andone scFv is linked to its C-terminus, the N-terminus of theimmunoglobulin CR is linked to a first VH domain, which is linked to afirst scFv linker, which is linked to a first VL domain, and the firstVH domain and the first VL domain form the first binding site; and theC-terminus of the immunoglobulin CR is linked to a second VH domain,which is linked to a second scFv linker, which is linked to a second VLdomain, and the second VH domain and the second VL domain form thesecond binding site and two such immunoglobulin CRs are dimerized orotherwise associated (e.g., by at least one bond, e.g., a disulfide bondor a van der Waals bond) to form a single tetravalent protein.

In preferred embodiments, the immunoglobulin CR is a humanimmunoglobulin CR, i.e., it essentially consists of an aa sequenceobtained from the human immunoglobulin repertoire. The immunoglobulin CRmay be that of any immunoglobulin isotype, class or subclass. In oneembodiment, an immunoglobulin CR is an IgG CR, such as an IgG1, IgG2,IgG3 or IgG4 CR. In certain embodiments, the CR is a hybrid that is madeup of at least two different classes or subclasses or types ofimmunoglobulins. For example, an immunoglobulin CR may have one domainfrom IgG1 and one or more other domains from an IgG4 protein. As furtherdescribed herein, in certain embodiments, a domain (e.g., CH1, hinge,CH2 or CH3) within the immunoglobulin CR may be mostly from one isotypeof immunoglobulin, but may have one or more aa mutation(s) (e.g.,substitution, addition or deletion) e.g., to provide the mutatedimmunoglobulin CR an attribute from another type or class ofimmunoglobulin CR.

In certain embodiments, a PBA has an IgG-(scFv)₂ structure. Suchproteins comprise an IgG antibody having two first binding sites, towhich is linked an scFv having a second binding site, e.g., to each ofthe two C-termini of the IgG protein. An exemplary IgG-(scFv)₂ is anIgG1-(scFv)₂, wherein the IgG is an IgG1.

In certain embodiments, a PBA comprises a heavy chain having thestructure represented by scFv-Fc-scFv and the PBA may have the structure(scFv-Fc-scFv)₂. The Fc may be an Fc region comprising a hinge, a CH2and a CH3 domain. In certain embodiments, such proteins do not comprisea CH1 or a CL domain.

In one embodiment, a PBA comprises two identical heavy-light chain pairsthat form an IgG like molecule, wherein each pair comprises one bindingmoiety that is an anti-IGF-1R Fab and another binding moiety that is ananti-ErbB3 scFv and wherein the two binding moieties are connectedthrough an immunoglobulin CR, which comprises in N-terminal toC-terminal order a hinge domain, a CH2 domain and a CH3 domain. The scFvmay be linked to the CH3 domain via a linker In an exemplary embodiment,a PBA comprises two identical heavy chains that form a dimer and twoidentical light chains, wherein each light chain associates with a heavychain, and wherein each heavy chain comprises: a first VH domain that islinked at its C-terminus to the N-terminus of a CH1 domain, which CH1domain is linked at its C-terminus to the N-terminus of a hinge domain,which hinge domain is linked at its C-terminus to the N-terminus of aCH2 domain, which CH2 domain is linked at its C-terminus to theN-terminus of a CH3 domain, which CH3 domain is linked at its C-terminusto the N-terminus of a linker, which linker is linked at its C-terminusto the N-terminus of a second VH domain, which second VH domain islinked at its C-terminus to the N-terminus of an scFv linker, which scFvlinker is linked at its C-terminus to the N-terminus of a second VLdomain, which second VL domain associates with the second VH domain toform the second binding site; and wherein each light chains comprises afirst VL domain that is linked at its C-terminus to the N-terminus of aCL domain, wherein the first VH domain and the first VL domain form thefirst binding site. In one embodiment, the first binding site is ananti-IGF-1R binding site and the second binding site is an anti-ErbB3binding site. In another embodiment, the first binding site is ananti-ErbB3 binding site and the second binding site is an anti-IGF-1Rbinding site.

In certain embodiments, a PBA comprises an IGF-1R binding sitecomprising a VHCDR3 consisting of the consensus sequence of SEQ IDNO:304, and optionally a VHCDR1 and/or VHCDR2 consisting of theconsensus sequences of SEQ ID NOs:302 and 303, respectively (see FIG.1). In certain embodiments, the last (C-terminal) X aa of SEQ ID NO:304is not I. A PBA may also comprise an anti-IGF-1R binding site comprisinga VLCDR3 consisting of the consensus sequence of SEQ ID NO:307 or 308,and optionally either or both of a VLCDR1 and a VLCDR2 consisting of thesequences of SEQ ID NOs:305 and 306, respectively (see FIG. 2). Incertain embodiments, a PBA comprises an anti-IGF-1R binding sitecomprising a VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3consisting of the consensus sequences of SEQ ID NOs:302, 303, 304, 305,306, and 307 (or 308), respectively.

In certain embodiments, a PBA comprises an anti-ErbB3 binding sitecomprising a VHCDR3 consisting of the consensus sequence of SEQ IDNO:311, and optionally a VHCDR1 and/or a VHCDR2 consisting of thesequences of SEQ ID NOs:309 and 310, respectively (see FIG. 3). A PBAmay also comprise an anti-IGF-1R binding site comprising a VLCDR3consisting of the consensus sequence of SEQ ID NO:314 or 315, andoptionally a VLCDR1 and/or VLCDR2 consisting of the sequences of SEQ IDNOs:312 and 313, respectively (see FIG. 4). In certain embodiments, aPBA comprises an anti-IGF-1R binding site comprising a VHCDR1, VHCDR2,VHCDR3, VLCDR1, VLCDR2 and VLCDR3 consisting of the sequences of SEQ IDNOs:309, 310, 311, 312, 313, and 314 (or 315), respectively.

The sequences of SEQ ID NOs:302-315 are set forth below:

Anti-IGF-1R CDRs: VHCDR1 consensus sequence:  (SEQ ID NO: 302)GFX1FSX2YPMH  VHCDR2 consensus sequence:   (SEQ ID NO: 303)ISX1X2GGATX3YADSVKG VHCDR3 consensus sequence:  (SEQ ID NO: 304)DFYX1X2LTGNAFDX3  VLCDR1 sequence:  (SEQ ID NO: 305) RASQGISSYLA VLCDR2 consensus sequence:  (SEQ ID NO: 306) AX1STX2QS VLCDR3 consensus sequence:  (SEQ ID NO: 307) QQYX1X2X3PLT and(SEQ ID NO: 308) QQYWX1X2PLT Anti-ErbB3 CDRs: VHCDR1 sequence: (SEQ ID NO: 309) GFTFDDYAMH  VHCDR2 consensus sequence: (SEQ ID NO: 310) ISWX1SGSX2GYADSVKG  VHCDR3 consensus sequence: (SEQ ID NO: 311) DLGX1X2QWX3X4GFDY  VLCDR1 sequence:  (SEQ ID NO: 312)QGDSLRSYYAS  VLCDR2 sequence:  (SEQ ID NO: 313) GKNNRPS VLCDR3 consensus sequence:  (SEQ ID NO: 314) X1SRDX2X3GX4X5WV     and(SEQ ID NO: 315) X1SRDX2PGX3X4WV

Each aa “X” followed by a numeral is a variable aa, which independentlyrepresents any aa, such as any aa located at a corresponding position inFIG. 1, 2, 3 or 4. Exemplary aas for X1-X2 of SEQ ID NO:302, X1-X2 ofSEQ ID NO:303 and X1-X3 of SEQ ID NO:304 are provided at thecorresponding positions in the aa sequences in FIG. 1. Exemplary aas forX1-X2 of SEQ ID NO:306, X1-X3 of SEQ ID NO:307 and X1-X2 of SEQ IDNO:308 are provided at the corresponding positions in the aa sequencesin FIG. 2. Exemplary aas for X1-X2 of SEQ ID NO:310 and X1-X4 of SEQ IDNO:311 are provided at the corresponding positions in the aa sequencesin FIG. 3. Exemplary aas for X1-X5 of SEQ ID NO:314 or X1-X4 of SEQ IDNO:315 are provided at the corresponding positions in the aa sequencesin FIG. 4.

An exemplary PBA comprises an anti-IGF-1R binding site comprising aVHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 consisting of thesequences of SEQ ID NOs:302, 303, 304, 305, 306, and 307 (or 308),respectively, and the anti-ErbB3 binding site comprising a VHCDR1,VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 consisting of the sequences ofSEQ ID NOs:309, 310, 311, 312, 313, and 314 (or 315), respectively.

Exemplary PBAs comprise one or more CDRs from one or more of the VRsprovided in FIGS. 1-4. In certain embodiments, an anti-IGF-1R bindingsite comprises 1, 2 or 3 CDRs of one of the VH domains of FIG. 1 and/or1, 2 or 3 CDRs of one of the VL domains of FIG. 2. For example, ananti-IGF-1R binding moiety may comprise CDR1, CDR2 and/or CDR3 from SEQID NO:11 and/or CDR1, CDR2 and/or CDR3 from SEQ ID NO:35 (CDRs of 16F).In certain embodiments, an anti-IGF-1R binding moiety comprises acombination of CDRs of FIGS. 1 and 2, with the proviso that (i.e.,wherein) (i) the binding moiety is not that of 16F, or (ii) 1, 2, 3, 4,5 or 6 of the CDRs of the anti-IGF-1R binding entities are not presentin the anti-IGF-1R binding entity of 16F, or (iii) the VH or VL domainsof the anti-IGF-1R binding entity is not identical to the correspondingVH or VL domains in 16F, respectively.

Exemplary PBAs comprise an anti-IGF-1R binding entity comprising a VHdomain comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of asequence in FIG. 1, e.g., one of SEQ ID Nos:8, 9, 10 and 11 (thelocation of these CDRs is shown in FIG. 1). PBAs may also comprise ananti-IGF-1R binding entity comprising a VL domain comprising the VLCDR1,VLCDR2 and VLCDR3 aa sequences of one of SEQ ID Nos:32, 33, 34 and 35(the location of these CDRs is shown in FIG. 2). In certain embodiments,PBAs comprise an anti-IGF-1R binding entity comprising a VH domaincomprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of one of SEQ IDNos:8, 9, 10 and 11 and a VL domain comprising the VLCDR1, VLCDR2 andVLCDR3 aa sequences of one of SEQ ID Nos:32, 33, 34 and 35. Inparticular embodiments, an anti-IGF-1R binding domain comprises a VHdomain comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of SEQ IDNo:8 and a VL domain comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of SEQ ID No:32. In particular embodiments, an anti-IGF-1Rbinding domain comprises a VH domain comprising the VHCDR1, VHCDR2 andVHCDR3 aa sequences of SEQ ID No:9 and a VL domain comprising theVLCDR1, VLCDR2 and VLCDR3 aa sequences of SEQ ID No:33. In particularembodiments, an anti-IGF-1R binding domain comprises a VH domaincomprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of SEQ ID No:10and a VL domain comprising the VLCDR1, VLCDR2 and VLCDR3 aa sequences ofSEQ ID No:34. In particular embodiments, an anti-IGF-1R binding domaincomprises a VH domain comprising the VHCDR1, VHCDR2 and VHCDR3 aasequences of SEQ ID No:11 and a VL domain comprising the VLCDR1, VLCDR2and VLCDR3 aa sequences of SEQ ID No:35. In particular embodiments, ananti-IGF-1R binding domain comprises a VH domain comprising the VHCDR1,VHCDR2 and VHCDR3 aa sequences of SEQ ID No:8 and a VL domain comprisingthe VLCDR1, VLCDR2 and VLCDR3 aa sequences of SEQ ID No:33. Inparticular embodiments, an anti-IGF-1R binding domain comprises a VHdomain comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of SEQ IDNo:10 and a VL domain comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of SEQ ID No:32.

In certain embodiments, an anti-ErbB3 binding site comprises 1, 2 or 3CDRs of one of the VH domains of FIG. 3 and/or 1, 2 or 3 CDRs of one ofthe VL domains of FIG. 4. For example, an anti-ErbB3 binding moiety maycomprise CDR1, CDR2 and/or CDR3 from SEQ ID NO:143 and/or CDR1, CDR2and/or CDR3 from SEQ ID NO:175 (CDRs of 16F). In certain embodiments, ananti-ErbB3 binding moiety comprises a combination of CDRs of FIGS. 1 and2, with the proviso that (i.e., wherein) (i) the binding moiety is notthat of 16F, or (ii) 1, 2, 3, 4, 5 or 6 of the CDRs of theanti-anti-ErbB3 binding entities are not present in the anti-ErbB3binding entity of 16F, or (iii) the VH or VL domains of the anti-ErbB3binding entity is not identical to the corresponding VH or VL domains in16F, respectively.

Exemplary PBAs comprise an anti-ErbB3 binding entity comprising a VHdomain comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of asequence in FIG. 3, e.g., one of SEQ ID Nos:134-143 (the location ofthese CDRs are provided in FIG. 3). PBAs may also comprise an anti-ErbB3binding entity comprising a VL domain comprising the VLCDR1, VLCDR2 andVLCDR3 aa sequences of one of SEQ ID Nos:166-175 (the location of theseCDRs are provided in FIG. 4). In certain embodiments, PBAs comprise ananti-ErbB3 binding entity comprising a VH domain comprising the VHCDR1,VHCDR2 and VHCDR3 aa sequences of one of SEQ ID Nos:134-143 and a VLdomain comprising the VLCDR1, VLCDR2 and VLCDR3 aa sequences of one ofSEQ ID NOs:166-175. In particular embodiments, an anti-ErbB3 bindingdomain comprises a VH domain comprising the VHCDR1, VHCDR2 and VHCDR3 aasequences of SEQ ID No:134 and a VL domain comprising the VLCDR1, VLCDR2and VLCDR3 aa sequences of SEQ ID No:166. In particular embodiments, ananti-ErbB3 binding domain comprises a VH domain comprising the VHCDR1,VHCDR2 and VHCDR3 aa sequences of SEQ ID No:135 and a VL domaincomprising the VLCDR1, VLCDR2 and VLCDR3 aa sequences of SEQ ID No:167.In particular embodiments, an anti-ErbB3 binding domain comprises a VHdomain comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of SEQ IDNo:136 and a VL domain comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of SEQ ID No:168. In particular embodiments, an anti-ErbB3binding domain comprises a VH domain comprising the VHCDR1, VHCDR2 andVHCDR3 aa sequences of SEQ ID No:137 and a VL domain comprising theVLCDR1, VLCDR2 and VLCDR3 aa sequences of SEQ ID No:169. In particularembodiments, an anti-ErbB3 binding domain comprises a VH domaincomprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of SEQ ID No:138and a VL domain comprising the VLCDR1, VLCDR2 and VLCDR3 aa sequences ofSEQ ID No:170. In particular embodiments, an anti-ErbB3 binding domaincomprises a VH domain comprising the VHCDR1, VHCDR2 and VHCDR3 aasequences of SEQ ID No:139 and a VL domain comprising the VLCDR1, VLCDR2and VLCDR3 aa sequences of SEQ ID No:171. In particular embodiments, ananti-ErbB3 binding domain comprises a VH domain comprising the VHCDR1,VHCDR2 and VHCDR3 aa sequences of SEQ ID No:140 and a VL domaincomprising the VLCDR1, VLCDR2 and VLCDR3 aa sequences of SEQ ID No:172.In particular embodiments, an anti-ErbB3 binding domain comprises a VHdomain comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of SEQ IDNo:141 and a VL domain comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of SEQ ID No:173. In particular embodiments, an anti-ErbB3binding domain comprises a VH domain comprising the VHCDR1, VHCDR2 andVHCDR3 aa sequences of SEQ ID No:142 and a VL domain comprising theVLCDR1, VLCDR2 and VLCDR3 aa sequences of SEQ ID No:174. In particularembodiments, an anti-ErbB3 binding domain comprises a VH domaincomprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of SEQ ID No:143and a VL domain comprising the VLCDR1, VLCDR2 and VLCDR3 aa sequences ofSEQ ID No:175. In particular embodiments, an anti-ErbB3 binding domaincomprises a VH domain comprising the VHCDR1, VHCDR2 and VHCDR3 aasequences of SEQ ID No:136 and a VL domain comprising the VLCDR1, VLCDR2and VLCDR3 aa sequences of SEQ ID No:169.

Binding sites may also comprise one or more CDRs of the VRs of FIGS.1-4, wherein 1, 2 or 3 aas have been changed, e.g., substituted, addedor deleted, provided that the binding sites are still able to bindspecifically to their target.

In certain embodiments, an anti-IGF-1R binding site comprises a VHdomain comprising the following consensus sequence:

(SEQ ID NO: 1; the CDRs are underlined)EVQLLQSGGGLVQPGGSLRLSCAASGFX1FSX2YPMHWVRQAPGKGLEWVX3SISX4X5GGATX6YADSVKGRFTISRDNSKNTLYLQMNSLRX7EDTAVYYCAKDFYX8X9LTGNAFDX10WGQGTX 11VTVSSThis consensus sequence was obtained by aligning the VH sequences of 24high affinity anti-IGF-1R binding sites. The alignment is shown in FIG.1.

In certain embodiments, each of aas X1-X11 of SEQ ID NO:1 independentlyrepresents any aa. In other embodiments, each of aas X1-X11 of SEQ IDNO:1 independently represents any aa set forth at those positions in anyof the sequences in FIG. 1. In one such embodiment, X1 is T, X2 is V, X3is S, X4 is S, X5 is S, X6 is R, X7 is A, X8 is D, X9 is I, X10 is I andX11 is T (SF heavy chain 16F; SEQ ID NO:11). Exemplary IGF-1R VHsequences are set forth as SEQ ID NOs:8-31.

In certain embodiments, a VH domain of an anti-IGF-1R binding sitecomprises the consensus sequence of SEQ ID NO:1 with the proviso that(i.e., wherein) the sequence is not that of SF heavy chain 16F, e.g., bydiffering from it in at least one aa. In certain embodiments, a VHdomain of an anti-IGF-1R binding site comprises the consensus sequenceof SEQ ID NO:1 with the proviso that (i.e., wherein) X1 is not T, X2 isnot V, X6 is not R, X8 is not D or X10 is not I. Exemplary anti-IGF-1RVH sequences are set forth as SEQ ID NOs:8-10 and 12-31.

In certain embodiments, an anti-IGF-1R binding site comprises a VLdomain comprising the following consensus sequence:

(SEQ ID NO: 2; the CDRs are underlined)DIQX1TQSPSSLSASX2GDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAX3STX4QSGVPSRFSGSGSGTX5FTLTISSLQPEDX6X7TYYCQQYX8X9X10PLTFGGGTKVEIK

This consensus sequence was obtained by aligning the VL sequences ofabout 100 high affinity anti-IGF-1R binding sites. The alignment isshown in FIG. 2.

In certain embodiments, each of aas X1-X10 of SEQ ID NO:2 independentlyrepresents any aa. In other embodiments, each of aas X1-X10 of SEQ IDNO:2 independently represents any aa set forth at those positions in anyof the sequences in FIG. 2. In one such embodiment, X1 is M, X2 is T, X3is A, X4 is L, X5 is D, X6 is F, X7 is A, X8 is F, X9 is T and X10 is F(SF kappa light chain 16F; SEQ ID NO:35). Exemplary IGF-1R VL sequencesare set forth as SEQ ID NOs:32-133.

In certain embodiments, a VL domain of an anti-IGF-1R binding sitecomprises the consensus sequence of SEQ ID NO:2, with the proviso that(i.e., wherein) the sequence is not that of SF light chain 16F, e.g., bydiffering from it in at least one aa. In certain embodiments, a VLdomain of an anti-IGF-1R binding site comprises the consensus sequenceof SEQ ID NO:2 with the proviso that (i.e., wherein) X2 is not T, X6 isnot F or X8 is not F. When X2 is not T, X6 is not F and/or X8 is not F,X2 may be an L, X6 may be an F and/or X8 may be an F, as of thefollowing consensus sequence:

(SEQ ID NO: 3; the CDRs are underlined)DIQX1TQSPSSLSASLGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAX2STX3QSGVPSRFSGSGSGTX4FTLTISSLQPEDSX5TYYCQQYWX6X7PLTFGGGTKVEIK

In certain embodiments, a VL domain of an anti-IGF-1R binding sitecomprises the consensus sequence of SEQ ID NO:3, wherein each of aasX1-X7 of SEQ ID NO:3 independently represents any aa. In otherembodiments, each of aas X1-X7 of SEQ ID NO:3 independently representsany aa set forth at those positions in any of the sequences in FIG. 2.Exemplary IGF-1R VL sequences are set forth as SEQ ID NOs:32-34 and36-133.

In certain embodiments, an anti-ErbB3 binding site comprises a VH domaincomprising the following consensus sequence:

(SEQ ID NO: 4; the CDRs are underlined)X1VQLVX2SGGGLVQPGX3SLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVX4GISWX5SGSX6GYADSVKGRFTISRDNAKNSLYLQMNSLRX7EDTAX8YYCARDLGX9X10QWX11X12GFDYWGQGTLVTVSS 

This consensus sequence was obtained by aligning the VH sequences of 32high affinity anti-IGF-1R binding sites. The alignment is shown in FIG.3.

In certain embodiments, each of aas X1-X12 of SEQ ID NO:4 independentlyrepresents any aa. In other embodiments, each of aas X1-X12 of SEQ IDNO:4 independently represents any aa set forth at those positions in anyof the sequences in FIG. 3. In one such embodiment, X1 is Q, X2 is Q, X3is G, X4 is A, X5 is N, X6 is I, X7 is P, X8 is V, X9 is Y, X10 is N,X11 is V and X12 is E (C8 heavy chain 16F; SEQ ID NO:143). ExemplaryErbB3 VH sequences are set forth as SEQ ID NOs:134-165.

In certain embodiments, a VH domain of an anti-ErbB3 binding sitecomprises the consensus sequence of SEQ ID NO:4, with the proviso that(i.e., wherein) the sequence is not that of C8 heavy chain 16F, e.g., bydiffering from it in at least one aa. In certain embodiments, a VHdomain of an anti-ErbB3 binding site comprises the consensus sequence ofSEQ ID NO:4 with the proviso that (i.e., wherein) aa X7 is not P or X8is not V. When X7 is not P and/or X8 is not V, X7 may be an A an/or X8may be an L, as of the following consensus sequence:

(SEQ ID NO: 5; the CDRs are underlined)X1VQLVX2SGGGLVQPGX3SLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVX4GISWX5SGSX6GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDLGX7X8QWX9X10GFDYWGQG TLVTVSS.

In certain embodiments, a VH domain of an anti-ErbB3 binding sitecomprises the consensus sequence of SEQ ID NO:5, wherein each of aasX1-X10 of SEQ ID NO:5 independently represents any aa. In otherembodiments, each of aas X1-X10 of SEQ ID NO:5 independently representsany aa set forth at those positions in any of the sequences in FIG. 3.Exemplary ErbB3 VH sequences are set forth as SEQ ID NOs:134-142 and144-165.

In certain embodiments, an anti-ErbB3 binding site comprises a VL domaincomprising the following consensus sequence:

(SEQ ID NO: 6; the CDRs are underlined).SX1ELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSX2SGNX3ASLTITGAQAEDEADYYCX4SRDX5X6GX7X8WVFGGGTKVTVX9G 

This consensus sequence was obtained by aligning the VL sequences of 35high affinity anti-IGF-1R binding sites. The alignment is shown in FIG.4.

In certain embodiments, each of aas X1-X9 of SEQ ID NO:6 independentlyrepresents any aa. In other embodiments, each of aas X1-X10 of SEQ IDNO:6 independently represents any aa set forth at those positions in anyof the sequences in FIG. 4. In one embodiment, X1 is Y, X2 is T, X3 isS, X4 is N, X5 is S, X6 is S, X7 is N, X8 is H, and X9 is L (C8 lambdalight chain 16F; SEQ ID NO:175). Exemplary ErbB3 VL sequences are setforth as SEQ ID NOs:166-200.

In certain embodiments, a VL domain of an anti-ErbB3 binding sitecomprises the consensus sequence of SEQ ID NO:6, with the proviso that(i.e., wherein) the sequence is not that of C8 lambda light chain 16F,e.g., by differing from it in at least one aa. In certain embodiments, aVL domain of an anti-ErbB3 binding site comprises the consensus sequenceof SEQ ID NO:6 with the proviso that (i.e., wherein) X6 is not S. WhenX6 is not S, X6 may be P, as of the following consensus sequence:

(SEQ ID NO: 7; the CDRs are underlined)SX1ELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSX2SGNX3ASLTITGAQAEDEADYYCX4SRDX5PGX6X7WVFGGGTKVTVX8G.

In certain embodiments, a VL domain of an anti-ErbB3 binding sitecomprises the consensus sequence of SEQ ID NO:7, each of aas X1-X8 ofSEQ ID NO:7 independently represents any aa. In other embodiments, eachof aas X1-X8 of SEQ ID NO:7 independently represents any aa set forth atthose positions in any of the sequences in FIG. 4. Exemplary ErbB3 VLsequences are set forth as SEQ ID NOs:166-174 and 176-200.

Exemplary anti-IGF-1R VH domains include the M57 VH domain (SEQ IDNO:8), M78 VH domain (SEQ ID NO:9), P4 VH domain (SEQ ID NO:10) and SFVH domain (SEQ ID NO:11). Exemplary anti-IGF-1R VL domains include theM57 VL domain (SEQ ID NO:32), M78 VL domain (SEQ ID NO:33), P4 VL domain(SEQ ID NO:34) and SF VL domain (SEQ ID NO:35).

Exemplary anti-ErbB3 VH domains include the B60 VH domain (SEQ IDNO:134), B72 VH domain (SEQ ID NO:135), M27 VH domain (SEQ ID NO:136),M7 VH domain (SEQ ID NO:137), P1 VH domain (SEQ ID NO:138), M27 VHdomain (SEQ ID NO:139), B69 VH domain (SEQ ID NO:140), P6 VH domain (SEQID NO:141), M1.3 VH domain (SEQ ID NO:142), and C8 VH domain (SEQ IDNO:143). Exemplary anti-ErbB3 VL domains include the B60 VL domain (SEQID NO:166), B72 VL domain (SEQ ID NO:167), M27 VL domain (SEQ IDNO:168), M7 VL domain (SEQ ID NO:169), P1 VL domain (SEQ ID NO:170), M27VL domain (SEQ ID NO:171), B69 VL domain (SEQ ID NO:172), P6 VH domain(SEQ ID NO:173), M1.3 VL domain (SEQ ID NO:174), and C8 VL domain (SEQID NO:175).

Binding sites may comprise a VH and a VL domain having the same modulename (e.g., “M57,” M78” and “P4”), i.e., which are the VH and VL domainsof an antibody of binding site thereof originally isolated, or they canbe mixed and matched. For example, anti-IGF-1R binding moieties maycomprise: (i) the VH domain of module M57 (SEQ ID NO:8) and the VLdomain of module M57 (SEQ ID NO:32); the VH domain of module M78 (SEQ IDNO:9) and the VL domain of module M78 (SEQ ID NO:33); (iii) the VHdomain of module P4 (SEQ ID NO:10) and the VL domain of module P4 (SEQID NO:34) or (iv) the VH domain of module SF (SEQ ID NO:11) and the VLdomain of module SF (SEQ ID NO:35). An anti-ErbB3 binding moieties maycomprise: (i) the VH domain of module B60 (SEQ ID NO:134) and the VLdomain of module B60 (SEQ ID NO:166); (ii) the VH domain of module B72(SEQ ID NO:135) and the VL domain of module B72 (SEQ ID NO:167); (iii)the VH domain of module M27 (SEQ ID NO:136) and the VL domain of moduleM27 (SEQ ID NO:168); (iv) the VH domain of module M7 (SEQ ID NO:137) andthe VL domain of module M7 (SEQ ID NO:169); (v) the VH domain of moduleP1 (SEQ ID NO:138) and the VL domain of module P1 (SEQ ID NO:170); (vi)the VH domain of module M27 (SEQ ID NO:139) and the VL domain of moduleM27 (SEQ ID NO:171); (vii) the VH domain of module B69 (SEQ ID NO:140)and the VL domain of module B69 (SEQ ID NO:172); (viii) the VH domain ofmodule P6 (SEQ ID NO:141) and the VL domain of module P6 (SEQ IDNO:173); (ix) the VH domain of module M1.3 (SEQ ID NO:142) and the VLdomain of module M1.3 (SEQ ID NO:174); and (x) the VH domain of moduleC8 (SEQ ID NO:143) and the VL domain of module C8 (SEQ ID NO:175).

Binding moieties of PBAs may also comprise mixed and matched VH and VLdomains, i.e., a binding moiety may comprise a VH domain from one moduleand a VL domain from another module. For example, an IGF-1R bindingmoiety may comprise the VH domain of module M57 (SEQ ID NO:8) and the VLdomain of module M78 (SEQ ID NO:33) or the VH domain of module P4 (SEQID NO:10) and the VL domain of module M57 (SEQ ID NO:32). An anti-ErbB3binding moiety may comprise the VH domain of module M27 (SEQ ID NO:136)and the VL domain of module M7 (SEQ ID NO:169; see, e.g., Example 7).Mixed chain binding moieties that bind with high affinity to theirtargets are preferred.

A PBA may also comprise aa sequences falling within more than one of theIGF-1R VH (SEQ ID NO:1), IGF-1R VL (SEQ ID NO:2), ErbB3 VH (SEQ ID NO:4)and ErbB3 VL (SEQ ID NO:6) consensus sequences described herein,provided that the PBA specifically binds to IGF-1R and ErbB3. Forexample, a PBA may comprise any of:

-   -   an IGF-1R VH domain comprising SEQ ID NO:1 and an IGF-1R VL        domain comprising SEQ ID NO:2;    -   an IGF-1R VH domain comprising SEQ ID NO:1 and an ErbB3 VH        domain comprising SEQ ID NO:4;    -   an IGF-1R VH domain comprising SEQ ID NO:1 and an ErbB3 VL        domain comprising SEQ ID NO:6;    -   an IGF-1R VH domain comprising SEQ ID NO:1, an IGF-1R VL domain        comprising SEQ ID NO:2, and an ErbB3 VH domain comprising SEQ ID        NO:4;    -   an IGF-1R VH domain comprising SEQ ID NO:1, an IGF-1R VL domain        comprising SEQ ID NO:2, and an ErbB3 VL domain comprising SEQ ID        NO:6;    -   an IGF-1R VH domain comprising SEQ ID NO:1, an IGF-1R VL domain        comprising SEQ ID NO:2, an ErbB3 VH domain comprising SEQ ID        NO:4, and an ErbB3 VL domain comprising SEQ ID NO:6;    -   an IGF-1R VL domain comprising SEQ ID NO:2 and an ErbB3 VH        domain comprising SEQ ID NO:4;    -   an IGF-1R VL domain comprising SEQ ID NO:2 and an ErbB3 VL        domain comprising SEQ ID NO:6;    -   an IGF-1R VL domain comprising SEQ ID NO:2, an ErbB3 VH domain        comprising SEQ ID NO:4, and an ErbB3 VL domain comprising SEQ ID        NO:6; or    -   an ErbB3 VH domain comprising SEQ ID NO:4 and an ErbB3 VL domain        comprising SEQ ID NO:6;    -   wherein the variable aas are independently any aa, or wherein        the variable aas are independently any of the aas set forth at        the corresponding position for each of these consensus sequences        of FIGS. 1-4, provided that the PBA binds specifically to IGF-1R        and to ErbB3.

A PBA may also comprise aa sequences falling within more than one of theIGF-1R VH (SEQ ID NO:1), IGF-1R VL (SEQ ID NO:2), ErbB3 VH (SEQ ID NO:4)and ErbB3 VL (SEQ ID NO:6) consensus sequences described herein,provided that the PBA specifically binds to IGF-1R and ErbB3, with theproviso that (i.e., wherein) the PBA is not 16F. For example a PBA maycomprise aa sequences falling within more than one of the IGF-1R VH (SEQID NO:1), IGF-1R VL (SEQ ID NO:3), ErbB3 VH (SEQ ID NO:5) and ErbB3 VL(SEQ ID NOs:7) consensus sequences described herein, provided that theyspecifically bind to IGF-1R and ErbB3. For example, a PBA may comprise:

-   -   an IGF-1R VH domain comprising SEQ ID NO:1 and an IGF-1R VL        domain comprising SEQ ID NO:3;    -   an IGF-1R VH domain comprising SEQ ID NO:1 and an ErbB3 VH        domain comprising SEQ ID NO:5;    -   an IGF-1R VH domain comprising SEQ ID NO:1 and an ErbB3 VL        domain comprising SEQ ID NO:7;    -   an IGF-1R VH domain comprising SEQ ID NO:1, an IGF-1R VL domain        comprising SEQ ID NO:3, and an ErbB3 VH domain comprising SEQ ID        NO:5;    -   an IGF-1R VH domain comprising SEQ ID NO:1, an IGF-1R VL domain        comprising SEQ ID NO:3, and an ErbB3 VL domain comprising SEQ ID        NO:7;    -   an IGF-1R VH domain comprising SEQ ID NO:1, an IGF-1R VL domain        comprising SEQ ID NO:3, an ErbB3 VH domain comprising SEQ ID        NO:5, and an ErbB3 VL domain comprising SEQ ID NO:7;    -   an IGF-1R VL domain comprising SEQ ID NO:3 and an ErbB3 VH        domain comprising SEQ ID NO:5;    -   an IGF-1R VL domain comprising SEQ ID NO:3 and an ErbB3 VL        domain comprising SEQ ID NO:7;    -   an IGF-1R VL domain comprising SEQ ID NO:3, an ErbB3 VH domain        comprising SEQ ID NO:5, and an ErbB3 VL domain comprising SEQ ID        NO:7; or    -   an ErbB3 VH domain comprising SEQ ID NO:5 and an ErbB3 VL domain        comprising SEQ ID NO:7;        wherein the variable aas are independently any aa, or wherein        the variable aas are independently any of the aas set forth at        the corresponding position for each of these consensus sequences        of FIGS. 1-4, provided that the PBA binds specifically to IGF-1R        and to ErbB3.

Exemplary PBAs comprise an anti-IGF-1R binding entity comprising a VHdomain comprising an aa sequence in FIG. 1, e.g., the aa sequence of oneof SEQ ID Nos:8, 9, 10 and 11. PBAs may also comprise an anti-IGF-1Rbinding entity comprising a VL domain comprising an aa sequence in FIG.2, e.g., the aa sequence of one of SEQ ID Nos:32, 33, 34 and 35. Incertain embodiments, PBAs comprise an anti-IGF-1R binding entitycomprising a VH domain comprising the aa sequence of one of SEQ IDNos:8, 9, 10 and 11 and a VL domain comprising the aa sequence of one ofSEQ ID Nos:32, 33, 34 and 35. In particular embodiments, an anti-IGF-1Rbinding domain comprises a VH domain comprising the aa sequence of SEQID No:8 and a VL domain comprising the aa sequence of SEQ ID No:32. Inparticular embodiments, an anti-IGF-1R binding domain comprises a VHdomain comprising the aa sequence of SEQ ID No:9 and a VL domaincomprising the aa sequence of SEQ ID No:33. In particular embodiments,an anti-IGF-1R binding domain comprises a VH domain comprising the aasequence of SEQ ID No:10 and a VL domain comprising the aa sequence ofSEQ ID No:34. In particular embodiments, an anti-IGF-1R binding domaincomprises a VH domain comprising the aa sequence of SEQ ID No:11 and aVL domain comprising the aa sequence of SEQ ID No:35. In particularembodiments, an anti-IGF-1R binding domain comprises a VH domaincomprising the aa sequence of SEQ ID No:8 and a VL domain comprising theaa sequence of SEQ ID No:33. In particular embodiments, an anti-IGF-1Rbinding domain comprises a VH domain comprising the aa sequence of SEQID No:10 and a VL domain comprising the aa sequence of SEQ ID No:32.

Exemplary PBAs comprise an anti-ErbB3 binding entity comprising a VHdomain comprising the aa sequence of one of SEQ ID Nos:134-143. PBAs mayalso comprise an anti-ErbB3 binding entity comprising a VL domaincomprising the aa sequence of one of SEQ ID Nos:166-175. In certainembodiments, PBAs comprise an anti-ErbB3 binding entity comprising a VHdomain comprising the aa sequence of one of SEQ ID Nos:134-143 and a VLdomain comprising the aa sequence of one of SEQ ID NOs:166-175. Inparticular embodiments, an anti-ErbB3 binding domain comprises a VHdomain comprising the aa sequence of SEQ ID No:134 and a VL domaincomprising the aa sequence of SEQ ID No:166. In particular embodiments,an anti-ErbB3 binding domain comprises a VH domain comprising the aasequence of SEQ ID No:135 and a VL domain comprising the aa sequence ofSEQ ID No:167. In particular embodiments, an anti-ErbB3 binding domaincomprises a VH domain comprising the aa sequence of SEQ ID No:136 and aVL domain comprising the aa sequence of SEQ ID No:168. In particularembodiments, an anti-ErbB3 binding domain comprises a VH domaincomprising the aa sequence of SEQ ID No:137 and a VL domain comprisingthe aa sequence of SEQ ID No:169. In particular embodiments, ananti-ErbB3 binding domain comprises a VH domain comprising the aasequence of SEQ ID No:138 and a VL domain comprising the aa sequence ofSEQ ID No:170. In particular embodiments, an anti-ErbB3 binding domaincomprises a VH domain comprising the aa sequence of SEQ ID No:139 and aVL domain comprising the aa sequence of SEQ ID No:171. In particularembodiments, an anti-ErbB3 binding domain comprises a VH domaincomprising the aa sequence of SEQ ID No:140 and a VL domain comprisingthe aa sequence of SEQ ID No:172. In particular embodiments, ananti-ErbB3 binding domain comprises a VH domain comprising the aasequence of SEQ ID No:141 and a VL domain comprising the aa sequence ofSEQ ID No:173. In particular embodiments, an anti-ErbB3 binding domaincomprises a VH domain comprising the aa sequence of SEQ ID No:142 and aVL domain comprising the aa sequence of SEQ ID No:174. In particularembodiments, an anti-ErbB3 binding domain comprises a VH domaincomprising the aa sequence of SEQ ID No:143 and a VL domain comprisingthe aa sequence of SEQ ID No:175. In particular embodiments, ananti-ErbB3 binding domain comprises a VH domain comprising the aasequence of SEQ ID No:136 and a VL domain comprising the aa sequence ofSEQ ID No:169.

In an exemplary embodiment, a PBA comprises a heavy chain comprising animmunoglobulin CR comprising, consisting essentially of, or consistingof the CH1 domain, hinge, CH2 domain and CH3 domain of an IgG1 (referredto as an “IgG1 CR”). Exemplary PBAs comprise an IgG1 CR and one or moreof the following aa sequences: an IGF-1R VH domain comprising aconsensus sequence of SEQ ID NO:1; an IGF-1R VL domain comprising aconsensus sequence of SEQ ID NO:2; an ErbB3 VH domain comprising theconsensus sequence of SEQ ID NO:4; and an ErbB3 VL domain comprising theconsensus sequence of SEQ ID NO:6, and comprise PBAs comprising heavyand light chain aa sequences of FIGS. 5A, 5B, 7A and 7B.

Other exemplary PBAs comprise a heavy chain comprising an IgG1 CR andone or more of the following aa sequences: an IGF-1R VH domaincomprising a consensus sequence of SEQ ID NO:1; an IGF-1R VL domaincomprising a consensus sequence of SEQ ID NO:3; an ErbB3 VH domaincomprising the consensus sequence of SEQ ID NO:5; and an ErbB3 VL domaincomprising the consensus sequence of SEQ ID NO:7, and comprise PBAscomprising heavy and light chain aa sequences of FIGS. 5A and 5B, butexcluding 16 heavy and light chains.

PBAs that comprise a carboxy terminus that would otherwise end with alysine or an arginine may have their carboxy terminal aa clipped by acarboxypeptidase. To avoid that, it may be beneficial to add one or moreaa at such a carboxy terminus. For example, when a protein wouldotherwise have “VEIK” at its carboxy terminus, 1, 2, 3, 4 5 or more aasmay be added to the carboxy terminus to prevent removal of the lysine.In certain embodiments, these additional aa may originate from the CLdomain. Thus, for example, in cases in which a PBA ends in ananti-IGF-1R VL sequence, which ends with “VEIK,” the aa “RT” may beadded at the carboxy terminus (see, e.g., P1-G1-P4; SEQ ID NO:268).

Anti-IGF-1R/ErbB3 PBAs may comprise a heavy chain comprising an aasequence selected from the group consisting of heavy chain fusions(hybrids): SF-G1-C8 (i.e., 16F; SEQ ID NO:210); SF-G1-P1 (SEQ IDNO:212); SF-G1-M1.3 (SEQ ID NO:214); SF-G1-M27 (SEQ ID NO:216); SF-G1-P6(SEQ ID NO:218); SF-G1-B69 (SEQ ID NO:220); P4-G1-C8 (SEQ ID NO:222);P4-G1-P1 (SEQ ID NO:224); P4-G1-M1.3 (SEQ ID NO:226); P4-G1-M27 (SEQ IDNO:228); P4-G1-P6 (SEQ ID NO:230); P4-G1-B69 (SEQ ID NO:232); M78-G1-C8(SEQ ID NO:234); M78-G1-P1 (SEQ ID NO:236); M78-G1-M1.3 (SEQ ID NO:238);M78-G1-M27 (SEQ ID NO:240); M78-G1-P6 (SEQ ID NO:242); M78-G1-B69 (SEQID NO:244); M57-G1-C8 (SEQ ID NO:246); M57-G1-P1 (SEQ ID NO:248);M57-G1-M1.3 (SEQ ID NO:250); M57-G1-M27 (SEQ ID NO:252); M57-G1-P6 (SEQID NO:254) and M57-G1-B69 (SEQ ID NO:256). Anti-IGF-1R/ErbB3 PBAs maycomprise a light chain comprising an aa sequence selected from the groupconsisting of Kappa light chains: SF (SEQ ID NO:202); P4 (SEQ IDNO:204); M78 (SEQ ID NO:206); and M57 (SEQ ID NO:208).

Anti-ErbB3/IGF-1R PBAs may comprise a heavy chain comprising an aasequence selected from the group consisting of heavy chain fusions(hybrids): P1-G1-P4 (SEQ ID NO:268); P1-G1-M57 (SEQ ID NO:270);P1-G1-M78 (SEQ ID NO:272); M27-G1-P4 (SEQ ID NO:274); M27-G1-M57 (SEQ IDNO:276); M27-G1-M78 (SEQ ID NO:278); M7-G1-P4 (SEQ ID NO:280); M7-G1-M57((SEQ ID NO:282); M7-G1-M78 (SEQ ID NO:284); B72-G1-P4 (SEQ ID NO:286);B72-G1-M57 (SEQ ID NO:288); B72-G1-M78 (SEQ ID NO:290); B60-G1-P4 (SEQID NO:292); B60-G1-M57 (SEQ ID NO:294); B60-G1-M78 (SEQ ID NO:296);B60-G2-M78 (SEQ ID NO:355) and M7-G2-M78 (SEQ ID NO:357).Anti-ErbB3/IGF-1R PBAs may comprise a light chain comprising an aasequence selected from the group consisting of lambda light chains: P1(SEQ ID NO:258), M27 (SEQ ID NO:260), M7 (SEQ ID NO:262), B72 (SEQ IDNO:264), and B60 (SEQ ID NO:266).

A heavy chain may be paired with a light chain that comprises a VLdomain of the same module as the VH domain of the heavy chain. However,heavy chains and light chains may also be mixed and matched, providedthat the binding sites retain high affinity binding to their target.

Exemplary IGF-1R+ErbB3 PBAs Comprising IgG1 CRs

The names of 38 exemplary IGF-1R+ErbB3 PBAs comprising IgG1 CRs are hereset forth below in Table 5, each of which names is followed by (inparentheses, in order) heavy chain SEQ ID NO and light chain SEQ ID NO.These IgG-like PBAs comprise two essentially identical heavy chains andtwo essentially identical light chains.

TABLE 5 Exemplary IGF-1R + ErbB3 PBAs comprising IgG1 CRs SF-G1-P1 (SEQID NO: 212 and SEQ ID NO: 202) SF-G1-M1.3 (SEQ ID NO: 214 and SEQ ID NO:202) SF-G1-M27 (SEQ ID NO: 216 and SEQ ID NO: 202) SF-G1-P6 (SEQ ID NO:218 and SEQ ID NO: 202) SF-G1-B69 (SEQ ID NO: 220 and SEQ ID NO: 202)P4-G1-C8 (SEQ ID NO: 222 and SEQ ID NO: 204) P4-G1-P1 (SEQ ID NO: 224and SEQ ID NO: 204) P4-G1-M1.3 (SEQ ID NO: 226 and SEQ ID NO: 204)P4-G1-M27 (SEQ ID NO: 228 and SEQ ID NO: 204) P4-G1-P6 (SEQ ID NO: 230and SEQ ID NO: 204) P4-G1-B69 (SEQ ID NO: 232 and SEQ ID NO: 204)M78-G1-C8 (SEQ ID NO: 234 and SEQ ID NO: 206) M78-G1-P1 (SEQ ID NO: 236and SEQ ID NO: 206) M78-G1-M1.3 (SEQ ID NO: 238 and SEQ ID NO: 206)M78-G1-M27 (SEQ ID NO: 240 and SEQ ID NO: 206) M78-G1-P6 (SEQ ID NO: 242and SEQ ID NO: 206) M78-G1-B69 (SEQ ID NO: 244 and SEQ ID NO: 206)M57-G1-C8 (SEQ ID NO: 246 and SEQ ID NO: 208) M57-G1-P1 (SEQ ID NO: 248and SEQ ID NO: 208) M57-G1-M1.3 (SEQ ID NO: 250 and SEQ ID NO: 208)M57-G1-M27 (SEQ ID NO: 252 and SEQ ID NO: 208) M57-G1-P6 (SEQ ID NO: 254and SEQ ID NO: 208) M57-G1-B69 (SEQ ID NO: 256 and SEQ ID NO: 208)P1-G1-P4 (SEQ ID NO: 268 and SEQ ID NO: 258) P1-G1-M57 (SEQ ID NO: 270and SEQ ID NO: 258) P1-G1-M78 (SEQ ID NO: 272 and SEQ ID NO: 258)M27-G1-P4 (SEQ ID NO: 274 and SEQ ID NO: 260) M27-G1-M57 (SEQ ID NO: 276and SEQ ID NO: 260) M27-G1-M78 (SEQ ID NO: 278 and SEQ ID NO: 260)M7-G1-P4 (SEQ ID NO: 280 and SEQ ID NO: 262) M7-G1-M57 ((SEQ ID NO: 282and SEQ ID NO: 262) M7-G1-M78 (SEQ ID NO: 284 and SEQ ID NO: 262)B72-G1-P4 (SEQ ID NO: 286 and SEQ ID NO: 264) B72-G1-M57 (SEQ ID NO: 288and SEQ ID NO: 264) B72-G1-M78 (SEQ ID NO: 290 and SEQ ID NO: 264)B60-G1-P4 (SEQ ID NO: 292 and SEQ ID NO: 266) B60-G1-M57 (SEQ ID NO: 294and SEQ ID NO: 266) B60-G1-M78 (SEQ ID NO: 296 and SEQ ID NO: 266).P4M-G1-M1.3 (SEQ ID NO: 376 and SEQ ID NO: 381) P4M-G1-C8 (SEQ ID NO:377 and SEQ ID NO: 381) P33M-G1-M1.3 (SEQ ID NO: 378 and SEQ ID NO: 380)P33M-G1-C8 (SEQ ID NO: 379 and SEQ ID NO: 380) P4M-G1-P6L (SEQ ID NO:389 and SEQ ID NO: 381) P33M-G1-P6L (SEQ ID NO: 390 and SEQ ID NO: 380)P1-G1-M76 (SEQ ID NO: 391 and SEQ ID NO: 258)

In an exemplary embodiment, a PBA comprises two identical heavy chainsand two identical light chains, wherein the sequence of each of theheavy chains comprises, consists essentially of, or consists of SEQ IDNO:210 or 300, and wherein the sequence of each of the light chainsdomains comprise, consist essentially of, or consist of SEQ ID NO:202 or298. PBAs may also comprise a heavy chain and/or a light chain thatcomprise an aa sequence that differs from SEQ ID NO:202 or 298 or SEQ IDNO:210 or 300, respectively, in exactly or in at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 50, 100, 200, or 300 aas, provided that thePBA has the desired biological characteristic(s), as further describedherein. In one embodiment, a PBA comprises an aa sequence that differsfrom SEQ ID NO:210 or 300 by the addition of a lysine (K) at the end ofthe CH3 domain, i.e., creating the sequence . . . SLSLSPGKGGGGS . . .(SEQ ID NO:301). A PBA may also comprise an aa sequence that differsfrom SEQ ID NO:210 or 300 by comprising one or more of the following aasubstitutions in the CH3 domain: S239D, N297Q, S298A, T299A, T299C,T299K, A330L, 1332E, E333A, K334A, E356D, M358L, N434A, N343K (EUnumbering; see FIG. 7A).

PBAs may also comprise two identical heavy chains and two identicallight chains, wherein the sequence of each of the heavy chains and ofeach of the light chains comprises, consists essentially of, or consistsof a sequence of FIG. 5A or 5B (i.e., any even SEQ ID-numbered sequenceselected from SEQ ID NOs:202-296). PBAs may also comprise a heavy chainand/or a light chain comprising an aa sequence that differs from asequence in FIG. 5A or 5B in exactly or in at most 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 50, 100, 200, or 300 aas, provided that thePBA has the desired biological characteristic(s), as further describedherein. The differences in aas may be aa deletions, additions orsubstitutions (e.g., conservative substitutions). For example, a PBA maycomprise an aa sequence that differs from an aa sequence of FIG. 5A or5B, by the addition (or deletion) of a lysine at the end of a CH3domain, and/or by comprising one or more of the following aasubstitutions in the CH3 domain: S239D, N297Q, S298A, T299A, T299C,T299K, A330L, 1332E, E333A, K334A, E356D, M358L, N434A, N343K (see FIG.7A). One or more aa differences may be present in one or both of the twoVH domains or in one or both of the two VL domains, e.g., in one or moreCDR or in one or more framework region (FR). One or more aa differencesmay also be present in one or more of the immunoglobulin CR domains,e.g., in the CH1 domain, the CL domain, the hinge, the CH2 domain and/orthe CH3 domain. Particular aa changes that may be made to animmunoglobulin CR, e.g., to change one or more characteristics of animmunoglobulin, are further described herein. Aa changes may also bepresent in the linker that connects the C-terminus of the CH3 domain tothe N-terminus of the scFv and/or the scFv linker that connects theC-terminus of the VH domain of the scFv to the N-terminus of the VLdomain of the scFv. Exemplary modifications that may be made to aconnecting linker and an scFv linker are discussed further herein.

The following PBAs may also be used:

-   -   PBAs comprising a heavy chain and/or a light chain comprising an        aa sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%,        98% or 99% identical to the aa sequence of the heavy chain        domain having SEQ ID NO:210 or 300 and/or the light chain domain        having SEQ ID NO:202 or 298 (SEQ ID NOs of 16F), wherein the        PBAs have the desired biological characteristic(s);    -   PBAs that are biosimilars or bioequivalents of a PBA consisting        of two heavy chain domains, each of which consists of SEQ ID        NO:210 or 300 and two light chain domains, each of which        consists of SEQ ID NO:202 or 298;    -   PBAs comprising one or more domains, e.g., VH domain, VL domain,        CDR domain, FR domain, CH1 domain, CL domain, hinge, CH2 domain,        CH3 domain, linker, scFv VH domain, scFv linker, and scFv VL        domain comprising an aa sequence that is at least 70%, 75%, 80%,        85%, 90%, 95%, 97%, 98% or 99% identical to the aa sequence of        the corresponding domain(s) in SEQ ID NO:202, 210, 298 or 300;    -   PBAs comprising a heavy chain domain and/or a light chain domain        comprising an aa sequence that is at least 70%, 75%, 80%, 85%,        90%, 95%, 97%, 98% or 99% identical to the aa sequence of a        heavy chain domain of FIG. 5A or 5B and/or a light chain domain        of FIG. 5A or 5B, wherein the PBAs have the desired biological        characteristic(s);    -   PBAs that are biosimilars or bioequivalents of a PBA consisting        of two heavy chain domains, each of which comprises an aa        sequence of FIG. 5A or 5B and two light chain domains, each of        which comprises an aa sequence of FIG. 5A or 5B;    -   PBAs comprising one or more domains, e.g., VH domain, VL domain,        CDR domain, FR domain, CH1 domain, CL domain, hinge domain, CH2        domain, CH3 domain, linker domain, scFv VH domain, scFv linker        domain, and scFv VL domain comprising an aa sequence that is at        least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to        the aa sequence of the corresponding domain(s) in any one of the        aa sequences of FIG. 5A or 5B.

In certain embodiments, in which a PBA comprises an aa sequence thatdiffers from an aa sequence (such as an aa of FIG. 5 or 6) set forthherein, the PBA is not 16F (i.e., does not comprise two heavy chainsconsisting of SEQ ID NO:210 and two light chains consisting of SEQ IDNO:202).

In certain embodiments, a PBA comprises two heavy-light chain pairs thatassociate with each other, wherein each heavy-light chain pair comprisesan anti-IGF-1R binding site and an anti-ErbB3 binding site, and whereinthe heavy-light chain pairs differ from each other. The heavy-lightchain pairs may differ in one or more aas (e.g., in exactly or in atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or up to 100aas). For example, a first heavy-light chain pair may comprise a heavychain CR and a second heavy-light chain pair may comprise a second heavyCR, wherein the first and the second heavy chain CRs have aa differencesthat favor their association with each other (e.g., “knobs and holes”).

In certain embodiments, a polyvalent protein comprises more than fourbinding sites. For example, a hexavalent binding protein may comprise anIgG-(scFv)₂, i.e., a protein comprising two binding sites that are partof the IgG, one scFvs is attached to the C-terminus of the each of theCH3 domains of the IgG, and further comprising either another Fab orscFv, linked, e.g., through a linker, to the N-terminus of the IgGprotein or to the C-terminus of each of the scFvs. An octavalent bindingprotein may comprise the same structure as a hexavalent binding protein,further comprising two additional binding sites.

In certain embodiments, a PBA may comprise one binding site that isderived from a binding protein or antibody that is known in the art,such as those further described herein. For example, a PBA may compriseone or more CDRs from an anti-IGF-1R antibody selected from the groupconsisting of CP-751,871; IMC-A12; ANTI-IGF-1R Ab# A; BIIB-G11; and C06,whose heavy and light chain aa sequences are of FIG. 37. For example, ananti-IGF-1R binding site for use in a PBA may comprise a VHCDR3 and/orVLCDR3 domain and optionally the VHCDR1, VLCDR1, VHCDR2 and/or VLCDR2 ofany one of SEQ ID NOs:321-335. In certain embodiment, a PBA comprises ananti-IGF-1R binding site comprising 1, 2, 3, 4, 5, or 6 CDRs comprisingan aa sequence that differs from the corresponding one of one of SEQ IDNOs:321-335 by 1, 2 or 3 aa additions, deletions or substitutions,provided that the binding site binds specifically to its target(antigen). In certain embodiments, a PBA comprises an anti-IGF-1Rbinding site comprising 1, 2, 3, 4, 5 or 6 CDRs comprising an aasequence that is at least 70%, 80%, 90% or 95% identical or similar tothe corresponding CDR of one of SEQ ID NOs:321-335. In certainembodiments, a PBA comprises a VH and/or a VL chain comprising an aasequence of any one of SEQ ID NOs:321-335. In other embodiments, a PBAcomprises a VH and/or VL chain that comprises an aa sequence thatdiffers from any one of SEQ ID NOs:321-335 in at most 1, 2, 3, 4, 5, 10,15, 20, 25 or 30 aa deletions, additions or substitutions, or which isat least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to asequence in SEQ ID NOs:321-335, provided that the binding site bindsspecifically to its target. In certain embodiments, a PBA comprises abinding site comprising a light chain and/or a heavy chain comprising anaa sequence of SEQ ID NOs:321-335, or which differs from it in at most1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 aadeletions, additions or substitutions, or which is at least 70%, 80%,85%, 90%, 95%, 97%, 98%, or 99% identical to a sequence in SEQ IDNOs:321-335, provided that the binding site retains specific binding toits target. For example, a PBA may comprise (i) a VH domain and a VLdomain; (ii) a heavy chain and a light chain; or (iii) an scFvcomprising the VH and VL chains, from an antibody selected from thegroup of antibodies consisting of CP-751,871; IMC-A12; ANTI-IGF-1R Ab#A; BIIB-G11; and C06. Such a PBA may comprise an anti-ErbB3 binding sitethat is described herein.

A PBA may comprise one or more CDRs from a anti-ErbB3 antibody, e.g.,anti-ErbB3 Ab# A; H3 (U.S. Pat. No. 7,332,580), MM Ab#3; MM Ab#14; MMAb#17 or MM Ab#19, whose heavy and light chain aa sequences are of FIG.38. For example, an anti-ErbB3 binding site for use in a PBA maycomprise a VHCDR3 and/or VLCDR3 domain and optionally a VHCDR1, VLCDR1,VHCDR2 and/or VLCDR2 of any one of SEQ ID NOs:336-353. In certainembodiment, a PBA comprises an anti-ErbB3 binding site comprising 1, 2,3, 4, 5, or 6 CDRs comprising an aa sequence that differs from thecorresponding one of one of SEQ ID NOs:336-353 by 1, 2 or 3 aaadditions, deletions or substitutions, provided that the binding sitebinds specifically to its target. In certain embodiments, a PBAcomprises an anti-ErbB3 binding site comprising 1, 2, 3, 4, 5 or 6 CDRScomprising an aa sequence that is at least 70%, 80%, 90% or 95%identical or similar to the corresponding CDR of one of SEQ IDNOs:336-353. In certain embodiments, a PBA comprises a VH and/or a VLchain comprising an aa sequence of any one of SEQ ID NOs:336-353. Inother embodiments, a PBA comprises a VH and/or VL chain that comprisesan aa sequence that differs from any one of SEQ ID NOs:336-353 in atmost 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 aa deletions, additions orsubstitutions, provided that the binding site retains specific bindingto its target, or which is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%,or 99% identical to a sequence in SEQ ID NOs:336-353. In certainembodiments, a PBA comprises a binding site comprising a light chainand/or a heavy chain comprising an aa sequence of SEQ ID NOs:336-353, orwhich differs from it in at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40,50, 60, 70, 80, 90 or 100 aa deletions, additions or substitutions, orwhich is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical toa sequence in SEQ ID NOs:336-353, provided that the binding site bindsspecifically to its target. For example, a PBA may comprise (i) a VHdomain and a VL domain; (ii) a heavy chain and a light chain; or (iii)an scFv comprising the VH and VL chains, from an antibody selected fromthe group of antibodies consisting of anti-ErbB3 Ab# A; H3 (U.S. Pat.No. 7,332,585), MM Ab#3; MM Ab#14; MM Ab#17 and MM Ab#19. Yet otheranti-ErbB3 binding sites (or portions thereof, such as CDRs, V domainsor chains) that may be used are those from the anti-ErbB3 antibodies1B4C3 (cat #sc-23865, Santa Cruz Biotechnology) and 2D1D12 (U3 PharmaAG), both of which are described in, e.g., US Pat Pub No. 20040197332and are produced by hybridoma cell lines DSM ACC 2527 or DSM ACC 2517(deposited at DSMZ), AV-203 (SEQ ID NO:190 (heavy chain) and SEQ IDNO:206 (light chain) in WO 2011/136911, Aveo Pharmaceuticals) or 8B8(produced by ATCC® hybridoma #HB-12070™, and described in WO1997/035885) those described in U.S. Pat. No. 7,846,440, the monoclonalantibody Mab 205.10.2 (SEQ ID NO:8 (heavy chain) and SEQ ID NO:10 (lightchain) in US Pat Pub No. 20110171222, Roche Glycart), the murineanti-ErbB3 antibody described in US Pat Pub No. 20100310557 (TrellisBiosciences) or a bispecific anti-ErbB3/anti-EGFR antibody (e.g., SEQ IDNO:14 (heavy chain) and SEQ ID NO:13 (light chain), Genentech). SuchPBAs may comprise an anti-IGF-1R binding site that is described herein.

PBAs may also comprise a binding site that binds to the same epitope onhuman IGF-1R or human ErbB3 as the binding moieties provided herein,e.g., it may compete with binding moieties having sequences as of FIGS.5A and 5B. Binding moieties encompassed herein may also compete forbinding to antigen with a binding moiety described herein, e.g., it maycompete with binding moieties having sequences as of FIGS. 5A and 5B. Abinding protein comprising a binding moiety that competes with a bindingmoiety described herein for binding to a target antigen or epitope maybe a binding moiety that is capable of displacing the binding moietydescribed herein when added to an ELISA before or after the bindingmoiety described herein.

In certain embodiments, PBAs provided herein do not include PBAs thatare of PCT/US2010/052712, in other embodiments PBAs provided herein donot include variable domains of PBAs that are of PCT/US2010/052712.

Exemplary Immunoglobulin CRs

In certain embodiments, a PBA comprises two heavy chains, wherein eachcomprises an immunoglobulin CR. The polyvalent binding domain may alsocomprise two light chains, wherein each light chain comprises a CLdomain. The immunoglobulin CR may be from a human Ig, e.g., a humanIgG1, IgG2, IgG3 or IgG4, or from more than one isotype ofimmunoglobulin. For example, one domain may be from an IgG1, and otherdomains may be from other IgG isotypes. In certain embodiments, aportion of one domain is from one IgG isotype and the other domains arefrom another IgG isotype.

A heavy chain immunoglobulin CR, which may comprise one or more of a CH1domain, a hinge, a CH2 domain, a CH3 domain, and a CH4 domain, maycomprise or consist of an aa sequence that is at least 70%, 75%, 80%,85%, 90%, 95%, 97%, 98% or 99% identical to a heavy chain immunoglobulinCR in a naturally occurring or wild type constant domain of a particularIgG isotype, e.g., IgG1, or a CR of one of the aa sequences set forthherein. A CL domain may also comprise or consist of a an aa sequencethat is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identicalto a CL domain in a naturally occurring or wild type kappa or lambdalight chain or a CL domain in one of the aa sequences set forth herein.

A heavy chain immunoglobulin CR, which may comprise one or more of a CH1domain, a hinge, a CH2 domain, a CH3 domain, and a CH4 domain, maycomprise exactly or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-15, 16-20,21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 50-100 or more aasubstitutions, additions and/or deletions relative to the same heavychain immunoglobulin CR in a naturally occurring or wild type constantdomain of a particular IgG isotype, e.g., IgG1, or relative to a heavychain immunoglobulin CR set forth herein, e.g., in FIGS. 5-7. A CLdomain may comprise exactly or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 50-100 or moreaa substitutions, additions and/or deletions relative to a CL domain ina naturally occurring or wild type kappa or lambda light chain or a CLdomain set forth herein.

Each domain of a CR, i.e., a CH1 domain, hinge, CH2 domain, CH3 domainand CL domain may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more) aa substitutions, additions and/or deletions relative to anaturally occurring or wild-type constant domain of a particular IgGisotype, e.g., IgG1, or a constant domain set forth herein. Each domainof a CR, i.e., a CH1 domain, hinge, CH2 domain, CH3 domain and CL domainmay comprise an aa sequence that is at least 70%, 75%, 80%, 85%, 90%,95%, 97%, 98% or 99% identical to the same domain in a naturallyoccurring or wild type constant domain of a particular IgG isotype,e.g., IgG1, or a domain set forth herein.

Aa substitutions, additions or deletions may be spatially positionedrelative to each other by an interval of at least 1 aa position or more,for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 aa positions ormore. In certain embodiments, engineered aas are spatially positionedapart from each other by an interval of at least 5, 10, 15, 20, or 25 aapositions or more.

In certain embodiments, PBAs comprise a CR, e.g., an Fc region or domainthereof, comprising an aa sequence set forth herein. In certainembodiments, PBAs comprise a CR, e.g., an Fc region or domain thereof,comprising an aa sequence set forth herein, wherein 1 or more aas havebeen deleted, added or substituted, or comprising an aa sequence that isat least about 80%, 90%, 95%, 97%, 98% or 99% identical to a sequenceset forth herein. For example, aas 356 and 358 in the CH3 domain of SEQID NO:300 or any other aa sequence in FIGS. 5 and 6 may be substituted,e.g., E356D and M358L, to mirror the wild type IgG1 CH3 sequence. Anyconstant domain variant that represents any haplotype is alsoencompassed herein.

The effect of certain aa changes to a constant domain can be determinedas further described herein or as known in the art.

Replacements of aa residues in the Fc portion to alter antibody effectorfunction are known in the art (U.S. Pat. Nos. 5,648,260 and 5,624,821).The Fc portion of an antibody mediates several important effectorfunctions e.g., cytokine induction, ADCC, phagocytosis, complementdependent cytotoxicity (CDC) and half-life/clearance rate of antibodyand antigen-antibody complexes. In some cases these effector functionsare desirable for a therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to FcγRs and complement CIq,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one aa residue is replaced in the CR of theantibody, for example the Fc region of the antibody, such that effectorfunctions of the antibody are altered. The dimerization of two identicalheavy chains of an immunoglobulin is mediated by the dimerization of CH3domains and is stabilized by the disulfide bonds within the hingeregion.

In one embodiment, a PBA retains one or more of, and preferably all ofthe following attributes: antibody-dependent cellular cytotoxicity(ADCC) and antibody-dependent cellular phagocytosis (ADCP) that inhumans are determined by interactions with activating FcγRI, FcγRIIa/c,FcγRIIIa and inhibitory FcγRIIb receptors; complement-dependentcytotoxicity (CDC) that is triggered by antibody binding to thecomponents of the complement system; and long half-life that is mediatedvia active recycling by the neonatal Fc receptor (FcRn). All of thesefunctions can be tuned to optimize the effectiveness of an anti-cancertherapy and are preferably retained in a PBA.

Certain aa modifications may be made to an immunoglobulin CR to reduceor increase the naturally biological activities of the constant domains,such as those set forth above. Accordingly, in certain embodiments, aconstant immunoglobulin region comprises an aa substitution, deletion oraddition at an aa position that is within the “15 Angstrom Contact Zone”of an Fc. The 15 Angstrom Zone includes residues located at EU positions243 to 261, 275 to 280, 282-293, 302 to 319, 336 to 348, 367, 369, 372to 389, 391, 393, 408, and 424-440 of a full-length, wild-type Fcmoiety.

In certain embodiments, a binding protein (e.g., a PBA, an anti-IgG-1Rbinding site and an anti-ErbB3 binding site) comprises an aa change(e.g., an aa substitution, addition or deletion) in an Fc domain thatalters one or more antigen-independent effector functions of the domain,e.g., the circulating half-life of a protein comprising the domain.Exemplary antibodies exhibit either increased or decreased binding toFcRn when compared to antibodies lacking such aa changes and, therefore,have an increased or decreased half-life in serum, respectively.Antibodies comprising Fc variants with improved affinity for FcRn areanticipated to have longer serum half-lives, whereas those comprising Fcvariants with decreased FcRn binding affinity are expected to haveshorter half-lives. In one embodiment, a binding protein with alteredFcRn binding comprises at least one Fc domain having one or more aachanges within the “FcRn binding loop” of an Fc domain. The FcRn bindingloop is comprised of aa residues 280-299 (EU) of a wild-type,full-length, Fc. In other embodiments, a binding protein having alteredFcRn binding affinity comprises at least one Fc domain having one ormore aa substitutions within the 15 Å FcRn “contact zone.” The term 15 ÅFcRn “contact zone” includes residues at the following positions of awild-type, full-length Fc domain: 243-261, 275-280, 282-293, 302-319,336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU). In certainembodiments, a binding protein having altered FcRn binding affinitycomprises at least one Fc domain (e.g., one or two Fc moieties) havingone or more aa changes at an aa position corresponding to any one of thefollowing EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342,376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplaryaa changes that alter FcRn binding activity are disclosed inInternational PCT Publication No. WO05/047327.

In some embodiments, a binding protein comprises an Fc variantcomprising an aa change that alters the antigen-dependent effectorfunctions of the polypeptide, in particular ADCC or complementactivation, e.g., as compared to a wild type Fc region. In exemplaryembodiment, said antibodies exhibit altered binding to an Fc gammareceptor (e.g., CD16). Such antibodies exhibit either increased ordecreased binding to FcγRs when compared to wild type polypeptides and,therefore, mediate enhanced or reduced effector function, respectively.Fc variants with improved affinity for FcγRs are anticipated to enhanceeffector function, and such proteins may have useful applications inmethods of treating mammals where target molecule destruction isdesired, e.g., in tumor therapy. In contrast, Fc variants with decreasedFcγR binding affinity are expected to reduce effector function. In oneembodiment, a binding protein comprises at least one alteredantigen-dependent effector function selected from the group consistingof opsonization, phagocytosis, complement dependent cytotoxicity,antigen-dependent cellular cytotoxicity (ADCC), or effector cellmodulation as compared to a binding protein comprising a wild type Fcregion.

In one embodiment a binding protein exhibits altered binding to anactivating FcγR (e.g., FcγRI, FcγRIIa, or FcγRIIIa). In anotherembodiment, a binding protein exhibits altered binding affinity to aninhibitory FcγR (e.g., FcγRIIb). In other embodiments, a binding proteinhaving increased FcγR binding affinity (e.g., increased FcγRIIIa bindingaffinity) comprises at least one Fc domain having an aa change at an aaposition corresponding to one or more of the following positions: 239,268, 298, 332, 334, and 378 (EU). In certain embodiments, a bindingprotein having decreased FcγR binding affinity (e.g., decreased FcγRI,FcγRII, or FcγRIIIa binding affinity) comprises at least one Fc domainhaving an aa substitution at an aa position corresponding to one or moreof the following positions: 234, 236, 239, 241, 251, 252, 261, 265, 268,293, 294, 296, 298, 299, 301, 326, 328, 332, 334, 338, 376, 378, and 435(EU).

In certain embodiments, a binding protein having increased complementbinding affinity (e.g., increased C1q binding affinity) comprises an Fcdomain having an aa change at an aa position corresponding to one ormore of the following positions: 251, 334, 378, and 435 (EU). In certainembodiments, a binding protein having decreased complement bindingaffinity (e.g., decreased C1q binding affinity) comprises an Fc domainhaving an aa substitution at an aa position corresponding to one or moreof the following positions: 239, 294, 296, 301, 328, 333, and 376 (EU).Exemplary aa changes that alter FcγR or complement binding activity aredisclosed in International PCT Publication No. WO05/063815. In certainembodiments, a binding protein may comprise one or more of the followingspecific Fc region substitutions: S239D, S239E, M252T, H268D, H268E,1332D, 1332E, N434A, and N434K (EU).

A binding protein may also comprise an aa substitution that alters theglycosylation of the binding protein. For example, an immunoglobulin CRof a binding protein may comprise an Fc domain having a mutation leadingto reduced glycosylation (e.g., N- or O-linked glycosylation) or maycomprise an altered glycoform of the wild-type Fc domain (e.g., a lowfucose or fucose-free glycan). An “engineered glycoform” refers to acarbohydrate composition that is covalently attached to an Fc region,wherein said carbohydrate composition differs chemically from that of aparent Fc region. Engineered glycoforms may be useful for a variety ofpurposes, including but not limited to enhancing or reducing effectorfunction. Engineered glycoforms may be generated by a variety of methodsknown in the art (U.S. Pat. No. 6,602,684; US Pat Pub No. 2010-0255013;US Pat Pub No. 20030003097; WO 00/61739A1; WO 01/29246A1; WO 02/31140A1;WO 02/30954A1); (Potelligent™ technology (Biowa, Inc., Princeton, N.J.);and GlycoMAb™ glycosylation engineering technology (GlycartBiotechnology AG, Zurich, Switzerland). Many of these techniques arebased on controlling the level of fucosylated and/or bisectingoligosaccharides that are covalently attached to the Fc region, forexample by expressing an Fc polypeptide in various organisms or celllines, engineered or otherwise (for example Lec-13 CHO cells or rathybridoma YB2/0 cells), by regulating enzymes involved in theglycosylation pathway (for example FUT8 [a1,6-fucosyltranserase] and/or(31-4-N-acetylglucosaminyl, transferase III [GnTIll]), or by modifyingcarbohydrate (s) after the Fc polypeptide has been expressed.

In exemplary embodiments, an aa change, e.g., an aa substitution resultsin an Fc region comprising reduced glycosylation of the N-linked glycannormally found at aa position 297 (EU). The Fc region may also comprisea low fucose or fucose free glycan at aa position 297 (EU). In certainembodiments, the binding protein has an aa substitution near or within aglycosylation motif, for example, an N-linked glycosylation motif thatcontains the aa sequence NXT or NXS. In a particular embodiment, abinding protein comprises an aa substitution at an aa positioncorresponding to 297 or 299 of Fc (EU). Exemplary aa substitutions thatreduce or alter glycosylation are disclosed in International PCTPublication No. WO05/018572 and US Pat Pub No. 20070111281.

In other embodiments, a binding protein comprises at least one Fc domainhaving one or more engineered cysteine residues or analog thereof thatare located at the solvent-exposed surface. Preferably the engineeredcysteine residue or analog thereof does not interfere with the desiredbiological activity of the binding protein. For example, it may bedesirable that the alteration does not interfere with the ability of theFc to bind to Fc receptors (e.g., FcγRI, FcγRII, or FcγRIII) orcomplement proteins (e.g., C1q), or to trigger immune effector function(e.g., antibody-dependent cytotoxicity (ADCC), phagocytosis, orcomplement-dependent cytotoxicity (CDCC)). In certain embodiments, theantibodies comprise an Fc domain comprising at least one engineered freecysteine residue or analog thereof that is substantially free ofdisulfide bonding with a second cysteine residue. The antibodies maycomprise an Fc domain having engineered cysteine residues or analogsthereof at one or more of the following positions in the CH3 domain:349-371, 390, 392, 394-423, 441-446, and 446b (EU). The antibodies maycomprise an Fc variant having engineered cysteine residues or analogsthereof at any one of the following positions: 350, 355, 359, 360, 361,389, 413, 415, 418, 422, 441, 443, and EU position 446b.

Desired effector functions may also be obtained by choosing an Fc from aparticular immunoglobulin class or subclass, or by combining particularregions from particular immuoglobulin classes or subclasses, e.g., IgG1,IgG2, etc. For example, since ADCC and CDC (through binding of IgG tothe FcγRs and C1q, respectively) is mediated by residues located in thehinge and CH2 domain, and since IgG4 essentially lacks effectorfunctions, an effectorless Fc may be constructed by combining the hingeand CH2 domain of IgG4 and the CH3 domain of IgG1. Fab-arm exchange inproteins comprising an IgG4 hinge may be reduced by the substitutionS228P in the hinge.

An immunoglobulin CR may also be modified by making changes, e.g., inthe CH3 domains, referred to in the art as “knobs-into-holes” anddescribed, e.g., in U.S. Pat. No. 7,183,076). In this strategy, the Fcportions of two heavy chains are engineered to give one a protruding“knob,” and the other a complementary “hole,” thereby favoring theassociation of the heavy chains.

Exemplary Linkers

Linkers may be used to connect two domains or regions together, e.g., avariable domain to a constant domain, a variable domain to a variabledomain and a constant domain to a constant domain. A linker connectingthe VH domain of an scFv to the VL domain of the scFv is referred to asan “scFv linker” A linker connecting an scFv to a constant domain, e.g.,a CH3 domain, is referred to as a “connecting linker”

Linkers are preferably of sufficient length to allow the proper foldingof the domains or regions being connected. For example, a linker may be1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or at least90-100 aas long.

In certain embodiments, a linker is biologically inert, e.g., mostlyincapable of inducing a biological response, e.g., an immune response.

A polypeptide linker may comprise or consist of a Gly-Ser linker A“Gly-Ser linker” refers to a peptide that consists of glycine and serineresidues. An exemplary Gly-Ser linker comprises an aa sequence havingthe formula (Gly₄Ser)_(n) (SEQ ID NO:395), wherein n is a positiveinteger (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20). For example, in certain embodiments, a connectinglinker comprises or consists of (Gly₄Ser)₃ (SEQ ID NO:396) or (Gly₄Ser)₄(SEQ ID NO:397) or (Gly₄Ser)₅ (SEQ ID NO:398). In certain embodiments,an scFv linker comprises or consists of (Gly₄Ser)₃ or (Gly₄Ser)₄ (SEQ IDNO:397) or (Gly₄Ser)₅ (SEQ ID NO:398). In addition to a (Gly₄Ser)_(n)sequence, a linker may also comprise one or more additional aas locatedN-terminally or C-terminally to the (Gly₄Ser)_(n) sequence. For example,an scFv linker may comprise 3 aas, e.g., AST located N-terminally to the(Gly₄Ser)_(n) (SEQ ID NO:399) sequence (see, e.g., in the heavy chainsequence of 16F of FIG. 7 and as SEQ ID NO:300. In that sequence, thescFv linker consists of the aa sequence AST (Gly₄Ser)₃ (SEQ ID NO:400).

Exemplary Biological Characteristics of PBAs

In certain embodiments, a PBA inhibits growth of tumor cells in vitro.As shown in Example 3(C) and in FIGS. 16 and 17, ananti-ErbB3/anti-IGF-1R IgG2(scfv)₂ PBA inhibited proliferation of twodifferent tumor cell lines in 2D cultures, whereas either binding sitealone did not significantly inhibit their proliferation. Thus, incertain embodiments, PBAs inhibit in vitro tumor cell proliferation morepotently (e.g., as measured by percent inhibition over a 6 day culture,e.g., the culture described in Example 3(C)), than either of the bindingsites alone. Proliferation by a PBA may be inhibited by at least 10%,20%, 30%, 40%, 50%, 60%, 70&, 80%, 90% or more, relative toproliferation of the cells in the absence of the PBA.

In certain embodiments, a PBA inhibits in vivo tumor cell proliferation.As shown in Example 3 (D) and FIGS. 18A to 20B, ananti-ErbB3/anti-IGF-1R IgG2(scfv)2 PBA inhibited proliferation of twodifferent tumor cell lines in a mouse model of cancer to a higher degreerelative to that by the individual binding sites. Thus, in certainembodiments, PBAs inhibit in vivo tumor cell proliferation more potently(e.g., as measured by comparing tumor size at the end of the experiment,e.g., that described in Example 3(D)), than either of the binding sitesalone. Tumor growth by a PBA may be inhibited by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or more, relative to tumor growth in theabsence of the PBA.

“Compared to either of the binding sites alone” refers to a comparisonwith one or the other of the binding sites of a PBA, when that bindingsite comprises the same VRs as the those of the binding site in the PBA,and is, e.g., in the form of an antibody, e.g., an IgG1 antibody (asshown, e.g., in the Examples).

In certain embodiments, a PBA inhibits signal transduction mediatedthrough either or both of IGF-1R and ErbB3. As shown in Examples 3(B), 4and 5(C), various PBAs inhibited signal transduction through IGF-1R andErbB3, as measured by inhibition of phosphorylation of IGF-1R, ErbB3 andAKT. The examples show that PBAs can inhibit signal transduction to asimilar or higher degree or extent relative to a prior art anti-IGF-1Rantibody (ANTI-IGF-1R Ab# A—SEQ ID NO:327 for the HC and SEQ ID NO:328for the LC) or anti-ErbB3 antibody (anti-ErbB3 Ab# A—SEQ ID NO:336 forHC and 337 for LC) or a combination thereof. In certain embodiments, thepercent reduction of levels of any one or combination of two or three ofpIGF-1R, pErbB3 and pAKT is comparable to (e.g., within 1%, 5%, or 10%),greater than (e.g., by 10%, 20%, 30%, 40% or 50%) or less than (e.g., by10%, 20%, 30%, 40%, 50%) that of ANTI-IGF-1R Ab# A or anti-ErbB3 Ab# A,or a combination thereof. In some embodiments, the inhibition ofphosphorylation (e.g., % inhibition) at the end of the experiment, e.g.,as described in the Examples, is comparable to, or higher, or lowerthan, that of a prior art anti-IGF-1R or anti-ErbB3 antibody orcombination thereof. In certain embodiments, a PBA inhibitsphosphorylation of IGF-1R, ErbB3 and/or AKT by at least 30%, 40%, 50%,60%, 70%, 80%, 85%, 90%, 95%, 98%, 99% or more, relative tophosphorylation in the absence of the PBA, when determined, e.g., at theend of the experiment, e.g., as of the Examples. Preferred PBAs inhibitIGF-1R and/or ErbB3 signal transduction, e.g., measured by inhibition ofphosphorylation of IGF-1R and ErbB3, nearly completely, e.g., by atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%.

Inhibition of a) ligand mediated phosphorylation of ErbB3, and b) IGF-1-or IGF-2-mediated phosphorylation of IGF-1R respectively can bedemonstrated by the ability of a PBA to reproducibly decrease the levelof phosphorylation of a) ErbB3 induced by an ErbB family ligand (e.g.,heregulin), b) IGF-1R induced by an IGF-1R ligand (i.e., IGF-1 orIGF-2), or c) AKT induced by an IGF-1R ligand or an ErbB3 ligand, eachrelative to the phosphorylation in control cells that are not contactedwith the PBA. The cell which expresses ErbB3 and/or IGF-1R can be anaturally occurring cell or a cell of a cell line or can berecombinantly produced by introducing nucleic acid encoding ErbB3 and/orIGF-1R into a host cell. In one embodiment, the PBA inhibits an ErbBfamily ligand mediated phosphorylation of ErbB3 by at least about 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99%, or more, as determined, for example, byWestern blotting followed by probing with an anti-phosphotyrosineantibody as described in the Examples infra. In another embodiment, thePBA inhibits IGF-1- or IGF-2-mediated phosphorylation of IGF-1R by atleast about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or more, as determined, forexample, by Western blotting followed by probing with ananti-phosphotyrosine antibody as described in the Examples infra.

PBAs may suppress heregulin-induced pAKT signaling in a cell, e.g., acancer cel, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100%. PBAs may suppress IGF-1-inducedpAKT signaling in a cell, e.g., a cancer cell, by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%.PBAs may suppress insulin-induced pAKT signaling in a cell, e.g., acancer cell, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100%. PBAs may suppress theIGF-2-induced pAKT signaling in a cell, e.g., a cancer cell, by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%%, 97%, 98%,99% or 100%. Inhibition of pAKT signaling may be determined as furtherdescribed herein, e.g., in the Examples.

PBAs may inhibit ligand induced phosphorylation of IGF-1R by at least70% or 80%, ligand induced phosphorylation of ErbB3 by at least 70% or80% and optionally ligand induced phosphorylation of AKT by at least 30%or 40%. PBAs may also inhibit ligand induced phosphorylation of IGF-1Rby at least 85%, ligand induced phosphorylation of ErbB3 by at least 85%and optionally ligand induced phosphorylation of AKT by at least 75%. Incertain embodiments, PBAs inhibit ligand induced phosphorylation ofIGF-1R by at least 50% and ligand induced phosphorylation of ErbB3 by atleast 90%, and optionally ligand induced phosphorylation of AKT by atleast 30%, 40%, 50%, 60%, 70%, 80% or 90%.

PBAs may also be defined by the EC50 (i.e. the concentration of PBA atwhich 50% of maximum inhibition is obtained) of their inhibition ofphosphorylation of one or more of IGF-1R, ErbB3 and AKT, which EC50s maybe determined as further described herein. For example, PBAs disclosedherein may inhibit phosphorylation of IGF-1R with an EC50 of 10⁻⁹ M,10⁻¹⁰ M or lower. They may inhibit phosphorylation of ErbB3 with an EC50of 10⁻⁹ M, 10⁻¹⁰ M or lower. They may inhibit phosphorylation of AKTwith an EC50 of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M or lower. Some PBAsdisclosed herein inhibit phosphorylation of IGF-1R by at least 80% or85% with an EC50 of 10⁻⁹ M, 10⁻¹⁰ M or lower; inhibit phosphorylation ofErbB3 by at least 80% or 85% with an EC50 of 10⁻⁹ M, 10⁻¹⁰ M or lower;and optionally inhibit phosphorylation of AKT by at least 55% or 65% or75% with an EC50 of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M or lower. In somecases, essentially complete blockage of either or both ofphosphorylation of IGF-1R and phosphorylation of ErbB3 will beobtainable with a PBA herein disclosed.

In some embodiments, a PBA provided herein binds to cells expressing oneor both of its targets (i.e., antigen(s) bound by the PBA) with an EC50of from about 0.02 nM or lower to about 10 nM, or with a Kd of about10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M, or lower; each asmeasured, e.g., by flow cytometry using such cells expressing one orboth of the target antigens of the PBA. In other embodiments, a PBAprovided herein binds to its target(s) (e.g., either or both of humanIGF-1R and human ErbB3) with a Kd of about 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹²M, or lower, as measured, e.g., by or bysurface Plasmon resonance using a BIAcore apparatus. For example, ananti-ErbB3/anti-IGF-1R IgG2(scfv)₂ PBA provided herein was shown to bindto ADRr and MCF7 cells with a Kd of 2.5 and 2.1 nM, respectively (seeExample 3A). Example 5(A) shows that several other PBAs bind to BxPC-3cells with an EC50 of about 2-5 nM. Example 5(B) shows that several PBAsbind to ErbB3 with an EC50 of about 0.2-0.4 nM. Other PBAs bind toBxPC-3 cells with an EC50 ranging from 0.02 nM (e.g., P4-G1-P6) to about1 nM or to about 2 nM.

In certain embodiments, a PBA binds an antigen (e.g., either ErbB3 orIGF-1R) with a dissociation constant (Kd) of 50 nM or less (i.e., abinding affinity at least as high as that indicated by a Kd of 50 nM)(e.g., a Kd of 40 nM or 30 nM or 20 nM or 10 nM or 1 nm, or 100 pM or 10pM or 1 pM or less). In a particular embodiment, a PBA binds an antigen(either ErbB3 or IGF-1R) with Kd of 8 nM or better (e.g., 7 nM, 6 nM, 5nM, 4 nM, 2 nM, 1.5 nM, 1.4 nM, 1.3 nM, 1 nM, 100 pM, 10 pM or 1 pM or0.1 pM or less). In other embodiments, the binding protein, bindingmoiety or binding site binds an antigen (e.g., ErbB3 or IGF-1R) with adissociation constant (Kd) of less than approximately 10⁻⁷M, such asless than about 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M or 10⁻¹² M or evenlower, and binds to the antigen with an affinity that is at least anorder of magnitude higher (i.e., a Kd value that is at least ten-foldlower) than its binding affinity for to a non-specific antigen (e.g.,KLH, BSA, or casein).

PBAs may inhibit the binding of a ligand to IGF-1R and/or ErbB3. Forexample, PBAs may inhibit binding of a ligand to IGF-1R and/or ErbB3 byat least 70%, 80%, 90%, 95%, 97%, 98% or 99%, as measured, e.g., oncells or in vitro. Certain PBAs inhibit the level of binding of a ligandto IGF-1R and/or ErbB3 when the ligand is added before or after the PBA.

In certain embodiments, a solution comprising PBAs at a concentration of0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mg/ml ormore (or ranges of concentrations between any of these two numbers)comprises more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%of PBAs in unaggregated form (referred to in this context as monomers)as determined e.g., by Size Exclusion Chromatography (SEC) e.g.,following, a stability test as described below. The percentage ofmonomers may be determined in a solution after one of the followingstability tests: a) incubation at 4° C. for 1, 2, 3, 4, 5, or 6 days, or1, 2, 3 or more weeks; b) incubation at room temperature for 1, 2, 3, 4,5, or 6 days, or 1, 2, 3 or more weeks; c) incubation at 37° C. for 1,2, 3, 4, 5, or 6 days, or 1, 2, 3 or more weeks; d) 1, 2, 3, 4 or 5cycles of freeze/thaw, and e) agitation, e.g., gentle agitation on theorbital shaker at room temperature, e.g., for 1, 2, 3, 4, 5 or morehours.

In certain embodiments, a PBA exhibits a stability after 1, 2, 3, 4 or 5days of incubation in serum at 37° C. of at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99%, relative to its stability at day 0,where the stability of a protein is determined by, e.g., measuring itsability to bind to one or more of its target antigens after incubation,as determined, e.g., by ELISA (see, e.g., Example 7).

In certain embodiments, a PBA has a melting temperature (Tm) asdetermined e.g., by Differential Scanning Fluorimetry (DSF) of at least50° C., 55° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C.,67° C., 68° C., 69° C. or 70° C., as described in the Examples.

In certain embodiments, a PBA effectively inhibits signal transductionthrough IGF-1R and/or ErbB3 when the ligand is present at a high or alow concentration. In certain embodiments, a PBA suppresses basalsignaling, e.g., suppresses the level of pAKT present in a cell in theabsence of ligand induction. A PBA may also down-regulate IGF-1R and/orErbB3, e.g., phosphorylated and/or non phosphorylated receptor, on thecell surface, e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or more, relative to a cell that was not exposed to the PBA. A PBAmay also inhibit insulin signaling in cells, e.g., by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or more, relative to a cell that wasnot exposed to the PBA.

PBAs may have a stability of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10days in mouse or human serum. PBAs may have a half-life of at least 10hours, 20 hours, 30 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70hours, 80 hours, 90 hours, 100 hours, 110 hours, 115 hours or more inCynomolgus monkeys when injected with either 5 or 25 mg/kg mice. Incertain embodiments, a PBA, has a half-life that is statisticallysignificantly longer, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 100% (i.e., 2 fold), 150% or 200% longer, in an organismthat is a mouse or a cynomolgus monkey than the half-life of anotherpolyvalent bispecific ab in the same organism, binding to the sameepitopes, wherein the orientation of antigen binding specificities isreversed between of the fab and of the scfv.

In certain embodiments, a PBA represses the protein level of mTOR or thelevel of phospho-mTOR, or reduces or inhibits mTOR activation (i.e.,reduces levels of pmTOR), e.g., by about 50%, by 2 fold, 3 fold, 4 fold,5 fold, or more relative to a monospecific anti-IGF-1R antibody thatbinds to the same epitope on IGF-1R as the PBA does.

PBAs may have a combination of two or more of the characteristics setforth herein. For example, a PBA may inhibit ligand inducedphosphorylation of IGF-1R by at least 80% and ligand inducedphosphorylation of ErbB3 by at least 80%, and also exhibit one or moreof the following characteristics: (i) a Tm, as determined by DSF, of atleast 60° C. or 65° C.; (ii) be at least 80%, 90% or 95% monomeric inPBS at 10 mg/mL after 5 days at room temperature; (iii) and have astability of at least 70%, 80% or 90% after 5 days of incubation inserum at 37° C. In certain embodiments, PBAs have a Tm of at least 60°C. and serum stability of at least 90%. In other embodiments, PBAs (i)inhibit growth of tumor cells, e.g., by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or more; (ii) inhibit signal transductionmediated through IGF-1R and/or ErbB3, e.g., by at least 70%, 80%, 90% ormore, (iii) are stable, e.g., be at least 80% monomeric in a solutionafter 1, 2, 3, 4, 5 or more days at 4° C., room temperature or 37° C.,and/or (iv) have a Tm, as determined by DSF, of at least 50° C., 55° C.,60° C., 65° C. or more; e.g., to a similar extent or more efficiently orpotently than either binding entity alone or together.

Standard assays may be used for determining the biological activity andcharacteristics of anti-IGF-1R+anti-ErbB3 PBAs. Exemplary assays for thefollowing tests are provided herein in the Examples: (a) assays fordetermining the binding affinity or Kd of a binding site to its target;(b) assays for determining the ability of a binding protein to bind to acell; (c) essays for determining the ability of a binding protein toinhibit signal transduction, by measuring inhibition of phosphorylationof IGF-1R, ErbB3 or AKT; (d) assays for determining the ability of a PBAto inhibit cell proliferation in vitro; (e) assays for determining theeffect of a PBA on tumor cells in vivo; and (f) assays for determiningthe stability of a PBA.

Monovalent and Divalent Monospecific Antibodies

Further provided herein are monovalent and bivalent monospecificantibodies that are either 1) bivalent IgG antibodies that can be madeby co-expression of at least one nucleic acid molecule encoding theheavy chain and at least one nucleic acid molecule, which may be thesame as or different from the molecule encoding the heavy chain, thatencodes the light chain of the antibody, or 2) monovalent single chainFv (scFv) antibodies that can be made by expression of at least onenucleic acid molecule encoding the scFv; each expressed in a suitableexpression system, as described herein, including commercially availableexpression systems and others that are well known in the art. Theseantibodies may be monoclonal.

Anti-IGF-1R Antibodies

Provided herein are anti-IGF-1R antibodies, which bind specifically tohuman IGF-1R. In certain embodiments, an IGF-1R binding proteincomprises a heavy chain and a light chain that associate with each otherto form a binding moiety, e.g., an antibody, or an antigen bindingdomain thereof. The description provided herein applies to anti-IGF-1Rantibodies, but also to anti-IGF-1R binding moieties that are comprisedin PBAs. Conversely, the description of anti-IGF-1R binding moietiesapplies to anti-IGF-1R antibodies or to antigen binding sites thereof.

In certain embodiments, an anti-IGF-1R binding protein comprises 1, 2,3, 4, 5, or 6 CDRs selected from the group consisting of a VHCDR1 aasequence that is encompassed in the consensus sequence of SEQ ID NO:302,a VHCDR2 aa sequence that is encompassed in the consensus sequence ofSEQ ID NO:303, a VHCDR3 aa sequence that is encompassed in the consensussequence of SEQ ID NO:304, the VLCDR1 aa sequence of SEQ ID NO:305, aVLCDR2 aa sequence that is encompassed in the consensus sequence of SEQID NO:306, and a VLCDR3 aa sequence that is encompassed in the consensussequence of SEQ ID NO:307 (or 308). For example, an anti-IGF-1R bindingprotein may comprise 1, 2, 3, 4, 5, or 6 CDRs selected from the groupconsisting of a VHCDR1, VHCDR2 and VHCDR3 aa sequences in one of the aasequences of FIG. 1, e.g., any one of SEQ ID NOs:8-31, and a VLCDR1,VLCDR2 and VLCDR3 aa sequence in one of the aa sequences of FIG. 2,e.g., one of SEQ ID NOs:32-133. In a particular embodiment, ananti-IGF-1R binding protein comprises a VH domain comprising 1, 2, or 3,4, 5, or 6 CDRs selected from the group consisting of a VHCDR1, VHCDR2or VHCDR3 aa sequence of SEQ ID NO:11 and a VLCDR1, VLCDR2 and VLCDR3 aasequence of SEQ ID NO:35 (CDRs of 16F). In certain embodiments, ananti-IGF-1R binding protein comprises a VH domain comprising 1, 2, 3, 4,5, or 6 CDRs selected from the group consisting of a VHCDR1, VHCDR2 andVHCDR3 aa sequences of a sequence in FIG. 1, e.g., SEQ ID NOs:8-10 and12-31, and a VLCDR1, VLCDR2 and VLCDR3 aa sequence that is of a sequencein FIG. 2, e.g., SEQ ID NOs:32-34 and 36-133.

In certain embodiments, an anti-IGF-1R binding protein comprises a VHdomain comprising an aa sequence that is encompassed by the consensussequence of SEQ ID NO:1 and/or a VL domain comprising an aa sequencethat is encompassed by the consensus sequence of SEQ ID NO:2. ExemplaryVH aa sequences are those of FIG. 1, e.g., SEQ ID NOs:8-31. Exemplary VLaa sequences are those of FIG. 2, e.g., SEQ ID NO:32-133. In oneembodiment, an anti-IGF-1R binding protein comprises a VH aa sequencecomprising SEQ ID NO:11 and/or a VL aa sequence comprising SEQ ID NO:35(variable domains of 16F).

In certain embodiments, an anti-IGF-1R binding protein comprises a VHdomain comprising an aa sequence that is encompassed by the consensussequence of SEQ ID NO:1 and/or a VL domain comprising an aa sequencethat is encompassed by the consensus sequence of SEQ ID NO:3. ExemplaryVH aa sequences are those set forth as SEQ ID NOs:8-10 and 12-31.Exemplary VL aa sequences are those set forth as SEQ ID NOs:32-34 and36-133.

Also provided herein are anti-IGF-1R antibodies that bind specificallyto IGF-1R, wherein the VH domain comprises an aa sequence that is atleast 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the aasequence of a VH aa sequence of FIG. 1, e.g., SEQ ID NOs:8-31, and/orwherein the VL domain comprises an aa sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the aa sequence ofa VL aa sequence of FIG. 2, e.g., SEQ ID NOs:32-133. In certainembodiments, the VH sequence of SEQ ID NO:11 and/or VL sequence of SEQID NO:35 is excluded.

Also provided herein are anti-IGF-1R antibodies that bind specificallyto IGF-1R, wherein the VH domain comprises an aa sequence that differsin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or in 1-5, 6-10, 11-15, 16-20, 21-25,26-30, 31-35, 36-40, 41-45, 46-50 or 50-100 aa substitutions, additionsor deletions from, an aa sequence of FIG. 1, e.g., SEQ ID NOs:8-31, andthe VL domain comprises an aa sequence that differs in 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or in 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40,41-45, 46-50 or 50-100 aa aa substitutions, additions or deletions from,an aa sequence of FIG. 2, e.g., SEQ ID NOs:32-133. In certainembodiments, the VH sequence of SEQ ID NO:11 and/or VL sequence of SEQID NO:35 is excluded.

Anti-IGF-1R antibodies may have the structure of an antibody, e.g., afull length antibody, or an antigen binding fragment thereof. Forexample, an anti-IGF-1R binding protein may comprise a heavy chain and alight chain, wherein the heavy chain comprises a in N- to C-terminalorder: a VH domain, a CH1 domain, a hinge, a CH2 domain, a CH3 domain,and optionally a CH4 domain, and wherein the light comprises in N— toC-terminal order: a VL domain and a CL domain. The constant domains arepreferably human and may be from IgG1, IgG2, IgG3, IgG4 or a combinationthereof. The constant domains may be naturally occurring sequences ormutated sequences, wherein one or more aa substitution, addition ordeletion has been made to the naturally occurring sequence(s).

An anti-IGF-1R binding protein may comprise a heavy chain comprising anaa sequence selected from the group consisting of SF heavy chain (SEQ IDNO:358); P4 heavy chain (SEQ ID NO:359); M78 heavy chain (SEQ ID NO:360)and M57 heavy chain (SEQ ID NO:361) (FIG. 6A). An anti-IGF-1R bindingprotein may also comprise a light chain comprising an aa sequenceselected from the group consisting of SF kappa light chain (SEQ IDNO:202); P4 kappa light chain (SEQ ID NO:204); M78 kappa light chain(SEQ ID NO:206); and M57 kappa light chain (SEQ ID NO:208) (FIG. 5A). Ananti-IGF-1R binding protein may comprise a heavy chain comprising an aasequence selected from the group consisting of SF heavy chain (SEQ IDNO:358); P4 heavy chain (SEQ ID NO:359); M78 heavy chain (SEQ ID NO:360)and M57 heavy chain (SEQ ID NO:361) and a light chain comprising an aasequence selected from the group consisting of SF kappa light chain (SEQID NO:202); P4 kappa light chain (SEQ ID NO:204); M78 kappa light chain(SEQ ID NO:206); and M57 kappa light chain (SEQ ID NO:208). In specificembodiments, IGF-1R antibodies comprise a heavy chain and a light chainhaving aa sequences having the same name, e.g., an SF heavy chain and anSF light chain, a P4 heavy chain and a P4 light chain, a M78 heavy chainand a M78 light chain, and a M57 heavy chain and a M57 light chain.However, heavy and light chains may also be mixed and matched. Forexample, an M57 heavy chain can be paired with a M7 light chain, and aP4 heavy chain can be paired with an M57 light chain.

Provided in particular are anti-IGF-1R IgG antibodies SF (heavy chainSEQ ID NO:358, kappa light chain SEQ ID NO:202); P4 (heavy chain SEQ IDNO:359, kappa light chain SEQ ID NO:204) M78 (heavy chain SEQ ID NO:360,kappa light chain SEQ ID NO:206) and M57 (heavy chain SEQ ID NO:361,kappa light chain SEQ ID NO:208); all of the IgG1 isotype.

In certain embodiments, an anti-IGF-1R binding protein comprises a heavychain comprising an aa sequence that is at least 70%, 75%, 80%, 85%,90%, 95%, 97%, 98% or 99% identical to an aa sequence of SEQ ID NOs:358,359, 360 and 361 and/or a light chain comprising an aa sequence that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to anaa sequence of SEQ ID NOs:202, 204, 206 and 208.

In other embodiments, an IGF-1R binding protein comprises a heavy chaincomprising an aa sequence that that differs in 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or in 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45,46-50 or 50-100 aa substitutions, additions or deletions from an aasequence of SEQ ID NOs:358, 359, 360 and 361 and/or a light chaincomprises an aa sequence that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or in 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50or 50-100 aa substitutions, additions or deletions from an aa sequenceselected from the group consisting of SEQ ID NOs:202, 204, 206 and 208.

Biological activities and characteristics of anti-ErbB3 antibodies maybe determined with assays, e.g., those described herein for PBAs.Anti-ErbB3 proteins may ligand-inhibit phosphorylation of ErbB3,proliferation of tumor cells and/or inhibition of tumor growth in vivo.

The anti-IGF-1R binding moieties may comprise, or be linked to, 1, 2, 3,4 or more other binding sites, which may be in the form of a Fab, anscFv or other form of binding site. For example, an anti-IGF-1R bindingprotein may comprise an anti-ErbB3 binding site, e.g., an anti-ErbB3scFv.

Anti-ErbB3 Antibodies

Also provided herein are anti-ErbB3 antibodies, which bind specificallyto human ErbB3. In certain embodiments, an ErbB3 binding proteincomprises a heavy chain and a light chain that associate with each otherand form a binding protein, e.g., an antibody, or an antigen bindingdomain thereof. The description provided below applies to anti-ErbB3antibodies, but also to anti-ErbB3 binding sites that are comprised inPBAs. Conversely, the description of anti-ErbB3 binding sites applies toanti-ErbB3 antibodies or antigen binding sites thereof.

In certain embodiments, an anti-ErbB3 binding protein comprises 1, 2, 3,4, 5, or 6 CDRs selected from the group consisting of the VHCDR1 aasequence of SEQ ID NO:309, a VHCDR2 aa sequence that is encompassed inthe consensus sequence of SEQ ID NO:310, a VHCDR3 aa sequence that isencompassed in the consensus sequence of SEQ ID NO:311, the VLCDR1 aasequence that is of SEQ ID NO:312, the VLCDR2 aa sequence that is of SEQID NO:313, and a VLCDR3 aa sequence that is encompassed in the consensussequence of SEQ ID NO:314 (or 315). For example, an anti-ErbB3 bindingprotein may comprise 1, 2, 3, 4, 5, or 6 CDRs selected from the groupconsisting of a VHCDR1, VHCDR2 and VHCDR3 aa sequence of a sequence inFIG. 3, e.g., in any one of SEQ ID NOs:134-165, and a VLCDR1, VLCDR2 andVLCDR3 aa sequence of a sequence in FIG. 4, e.g., in SEQ ID NOs:166-200.In a particular embodiment, an anti-ErbB3 binding protein comprises a VHdomain comprising 1, 2, or 3, 4, 5, or 6 CDRs selected from the groupconsisting of a VHCDR1, VHCDR2 or VHCDR3 aa sequence of SEQ ID NO:143and a VLCDR1, VLCDR2 and VLCDR3 aa sequence of SEQ ID NO:175 (CDRs of16F). In certain embodiments, an anti-ErbB3 binding protein comprises aVH domain comprising 1, 2, 3, 4, 5, or 6 CDRs selected from the groupconsisting of a VHCDR1, VHCDR2 or VHCDR3 aa sequence of FIG. 3, e.g.,any one of SEQ ID NOs:134-142 and 144-165, and a VLCDR1, VLCDR2 andVLCDR3 aa sequence that is of FIG. 4, e.g., any one of SEQ IDNOs:166-174 and 176-200.

In certain embodiments, an anti-ErbB3 binding protein comprises a VHdomain comprising an aa sequence that is encompassed by the consensussequence of SEQ ID NO:4 and/or a VL domain comprising an aa sequencethat is encompassed by the consensus sequence of SEQ ID NO:6. ExemplaryVH aa sequences are those of FIG. 3, e.g., SEQ ID NOs:134-165. ExemplaryVL aa sequences are those of Table 4, e.g., SEQ ID NO:166-200. In oneembodiment, an anti-ErbB3 binding protein comprises a VH aa sequencecomprising SEQ ID NO:143 and/or a VL aa sequence comprising SEQ IDNO:175 (variable domains of 16F).

In certain embodiments, an anti-ErbB3 binding protein comprises a VHdomain comprising an aa sequence that is encompassed by the consensussequence of SEQ ID NO:5 and/or a VL domain comprising an aa sequencethat is encompassed by the consensus sequence of SEQ ID NO:7. ExemplaryVH aa sequences are those set forth as SEQ ID NOs:134-142 and 145-165.Exemplary VL aa sequences are those set forth as SEQ ID NOs:166-174 and176-200.

Also provided herein are anti-ErbB3 antibodies that bind specifically toErbB3, wherein the VH domain comprises an aa sequence that is at least70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the aasequence of a VH aa sequence of FIG. 3, e.g., SEQ ID NOs:134-165, and/orwherein the VL domain comprises an aa sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the aa sequence ofa VL aa sequence of FIG. 4, e.g., SEQ ID NOs:166-200. In certainembodiments, the VH sequence of SEQ ID NO:143 and/or VL sequence of SEQID NO:175 is excluded.

Also provided herein are anti-ErbB3 antibodies that bind specifically toErbB3, wherein the VH domain comprises an aa sequence that differs in 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or less of from 1-5, 6-10, 11-15, 16-20,21-25, 26-30, 31-35, 36-40, 41-45, 46-50 or 50-100 aa substitutions,additions or deletions from, the aa sequence of FIG. 3, e.g., SEQ IDNOs:134-165, and the VL domain comprises an aa sequence that differs in1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or less of from 1-5, 6-10, 11-15, 16-20,21-25, 26-30, 31-35, 36-40, 41-45, 46-50 or 50-100 aa substitutions,additions or deletions from, the aa sequence of FIG. 4, e.g., SEQ IDNOs:166-200. In certain embodiments, the VH sequence of SEQ ID NO:143and/or VL sequence of SEQ ID NO:175 is excluded.

Anti-ErbB3 antibodies may have the structure of an antibody, e.g., aholo-antibody (full length antibody) or an antigen binding fragmentthereof. For example, an anti-ErbB3 binding protein may comprise a heavychain and a light chain, wherein the heavy chain comprises a in N— toC-terminal order: a VH domain, a CH1 domain, a hinge, a CH2 domain, aCH3 domain, and optionally a CH4 domain, and wherein the light comprisesin N— to C-terminal order: a VL domain and a CL domain. The constantdomains are preferably human and may be from IgG1, IgG2, IgG3, IgG4 or acombination thereof. The constant domains may be naturally occurringsequences or mutated sequences, wherein one or more aa substitution,addition or deletion has been made to the naturally occurringsequence(s).

An anti-ErbB3 binding protein may comprise a heavy chain comprising anaa sequence selected from the group consisting of P1 heavy chain (SEQ IDNO:362); M27 heavy chain (SEQ ID NO:363); M7 heavy chain (SEQ ID NO:364)B72 heavy chain (SEQ ID NO:365) and B60 (SEQ ID NO:366) (FIG. 5B). Ananti-ErbB3 binding protein may also comprise a light chain comprising anaa sequence selected from the group consisting of P1 lambda light chain(SEQ ID NO:258); M27 lambda light chain (SEQ ID NO:260); M7 lambda lightchain (SEQ ID NO:262); B72 lambda light chain (SEQ ID NO:264) and B60lambda light chain (SEQ ID NO:266) (FIG. 5B). An anti-ErbB3 bindingprotein may comprise a heavy chain comprising an aa sequence selectedfrom the group consisting of P1 heavy chain (SEQ ID NO:362); M27 heavychain (SEQ ID NO:363); M7 heavy chain (SEQ ID NO:364) B72 heavy chain(SEQ ID NO:365) and B60 (SEQ ID NO:366) and a light chain comprising anaa sequence selected from the group consisting of P1 lambda light chain(SEQ ID NO:258); M27 lambda light chain (SEQ ID NO:260); M7 lambda lightchain (SEQ ID NO:262); B72 lambda light chain (SEQ ID NO:264) and B60lambda light chain (SEQ ID NO:266). In specific embodiments, IGF-1Rantibodies comprise a heavy chain and a light chain having aa sequenceshaving the same name, e.g., an P1 heavy chain and an P1 light chain, aM27 heavy chain and a M27 light chain, a M7 heavy chain and M7 lightchain, a B72 heavy chain and a B72 light chain, and a B60 heavy chainand a B60 light chain. However, heavy and light chains may also be mixedand matched. For example, an M57 heavy chain can be paired with a M7light chain, and a P4 heavy chain can be paired with an M57 light chain.

Also provided are anti-ErbB3 antibodies P1 (heavy chain SEQ ID NO:362,lambda light chain SEQ ID NO:258); M27 (heavy chain SEQ ID NO:363,lambda light chain SEQ ID NO:260); M7 (heavy chain SEQ ID NO:364, lambdalight chain SEQ ID NO:262); B72 (heavy chain SEQ ID NO:365, lambda lightchain SEQ ID NO:264); and B60 (heavy chain SEQ ID NO:366, lambda lightchain SEQ ID NO:266); all of the IgG1 isotype.

In certain embodiments, an anti-ErbB3 binding protein comprises a heavychain comprising an aa sequence that is at least 70%, 75%, 80%, 85%,90%, 95%, 97%, 98% or 99% identical to an aa sequence of SEQ ID NOs:362,363, 364, 365 and 366 and/or a light chain comprising an aa sequencethat is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identicalto an aa sequence of SEQ ID NOs:258, 260, 262, 264 and 266.

In other embodiments, an ErbB3 binding protein comprises a heavy chaincomprising an aa sequence that that differs in 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or in 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45,46-50 or 50-100 aa substitutions, additions or deletions from an aasequence of SEQ ID NOs:362, 363, 364, 365 and 366 and/or a light chaincomprises an aa sequence that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or in 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50or 50-100 aa substitutions, additions or deletions from an aa sequenceselected from the group consisting of SEQ ID NOs:258, 260, 262, 264 and266.

Biological activities and characteristics of anti-ErbB3 antibodies maybe determined with assays, e.g., those described herein for PBAs.Anti-ErbB3 proteins may ligand-inhibit phosphorylation of ErbB3,proliferation of tumor cells and/or inhibition of tumor growth in vivo.

The anti-ErbB3 binding moieties may comprise, or be linked to, 1, 2, 3,4 or more other binding sites, which may be in the form of a Fab, anscFv or other form of binding site. For example, an anti-ErbB3 bindingprotein may comprise an anti-IGF-1R binding site, e.g., an anti-IGF-1RscFv.scFv Antibodies

Also provided are scFvs, e.g., isolated monoclonal scFvs. ExemplaryscFvs are anti-IGF-1R scFvs and anti-ErbB3 scFvs. Exemplary scFvs arepolypeptides comprising a VH domain and a VL domain that are linkedtogether by an scFv linker. The VH and VL chains of an scFv are joinedtogether by an scFv linker that is interposed between the VH and VLchains. scFv linkers may consist of a contiguous aa sequence of 10-30aa, such as 15 to 20 aa. Exemplary scFv linkers are Gly-Ser linkers (SEQID NO:399),

which may be (Gly₄Ser)_(n) (SEQ ID NO:401), wherein n is 1, 2, 3, 4, 5,6, 7, 8, 9 or 10. Preferred scFv linkers comprise (Gly₄Ser)₃ (SEQ IDNO:396) or (Gly₄Ser)₄ (SEQ ID NO:397). Other preferred scFv linkerscomprise 1-5 aa in addition to (Gly₄Ser)₃ (SEQ ID NO:396) or (Gly₄Ser)₄(SEQ ID NO:397), e.g., AST, and may comprise the following aa sequence:AST(Gly₄Ser)₃ (SEQ ID NO:400) or AST(Gly₄Ser)₄ (SEQ ID NO:402).

An anti-IGF-1R scFv antibody may comprise a VH domain comprising a setof three VHCDRs comprising VHCDR1, VHCDR2 and VHCDR3, and a VL domaincomprising a set of three VLCDRs comprising VLCDR1, VLCDR2 and VLCDR3,said CDRs comprising the aa sequences of SEQ ID NOs:302, 303 or 304,305, 306 or 307 (or 308), respectively, and wherein each CDR furthercomprises an amino terminus and a carboxy terminus, wherein the CDRs ofeach set of CDRs are arranged in the variable domain in a linear aminoto carboxy order of CDR1, CDR2 and CDR3, and wherein X aa in SEQ IDNOs:302, 304, 305, 306, 307 (or 308) represent variable aa, which may beany aa located in the corresponding position in FIG. 1 (for VH) and FIG.2 (for VL). Anti-IGF-1R scFvs may comprise a VHCDR1, VHCDR2 and VHCDR3of a VH domain consisting of an aa sequence of the group of VH aasequences in FIG. 1, e.g., consisting of SEQ ID NOs:8-31, and/or aVLCDR1, VLCDR2 and VLCDR3 of a VL domain consisting of an aa sequence ofthe group of VL aa sequences in FIG. 2, e.g., consisting of SEQ IDNOs:32-133. In certain embodiments, an anti-IGF-1R scFv does notcomprise all six CDRs of 16F or does not comprise either the VH domainof 16F and/or the VL domain of 16F. For example, an scFv comprises VHand VL aa sequences that differ from those in 16F in at least one aa.

Anti-IGF-1R scFv antibodies may comprise a VH domain comprising the aasequence of SEQ ID NO:1 and/or a VL domain comprising the aa sequenceset forth SEQ ID NO:2 (or 3), wherein the X aas in SEQ ID NOs:1, 2 and 3are variable aa which may be any aa at the corresponding position inFIG. 1 (for the VH domain) and FIG. 2 (for the VL domain). Anti-IGF-1RscFv antibodies may comprise a VH domain comprising an aa sequence ofFIG. 1, e.g., selected from the group consisting of SEQ ID NOs:8-31,and/or a VL domain comprising an aa sequence of FIG. 2, e.g., selectedfrom the group consisting of SEQ ID NOs:32-133.

Exemplary anti-IGF-1R scFvs comprise a VH domain consisting of, or atleast comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of, an aasequence selected from the group of VH aa sequences consisting of SEQ IDNos:8, 9, 10 and 11 (the location of these CDRs is shown in FIG. 1).Anti-IGF-1R scFvs may also comprise a VL domain consisting of, or atleast comprising the VLCDR1, VLCDR2 and VLCDR3 aa sequences of, an aasequence selected from the group of VL aa sequences consisting of SEQ IDNos:32, 33, 34 and 35 (the location of these CDRs is shown in FIG. 2).In certain embodiments, anti-IGF-1R scFvs comprise a VH domainconsisting of, or at least comprising the VHCDR1, VHCDR2 and VHCDR3 aasequences of, an aa sequence selected from the group of VH aa sequencesconsisting of SEQ ID Nos:8, 9, 10 and 11 and a VL domain consisting of,or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aa sequences of, anaa sequence selected from the group of VL aa sequences consisting of SEQID Nos:32, 33, 34 and 35. In particular embodiments, an anti-IGF-1R scFvcomprises a VH domain comprising the VH domain consisting of, or atleast comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ IDNo:8 and a VL domain consisting of, or at least comprising the VLCDR1,VLCDR2 and VLCDR3 aa sequences of, an aa sequence consisting of SEQ IDNo:32 (M57 module). In particular embodiments, an anti-IGF-1R scFvcomprises a VH domain comprising the VH domain consisting of, or atleast comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ IDNo:9 and a VL domain consisting of, or at least comprising the VLCDR1,VLCDR2 and VLCDR3 aa sequences of, an aa sequence consisting of SEQ IDNo:33 (module M78). In particular embodiments, an anti-IGF-1R scFvcomprises a VH domain comprising the VH domain consisting of, or atleast comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ IDNo:10 and a VL domain consisting of, or at least comprising the VLCDR1,VLCDR2 and VLCDR3 aa sequences of, an aa sequence consisting of SEQ IDNo:34 (module P4). In particular embodiments, an anti-IGF-1R scFvcomprises a VH domain comprising the VH domain consisting of, or atleast comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ IDNo:8 and a VL domain consisting of, or at least comprising the VLCDR1,VLCDR2 and VLCDR3 aa sequences of, an aa sequence consisting of SEQ IDNo:33 (module M57/M78). In particular embodiments, an anti-IGF-1R scFvcomprises a VH domain comprising the VH domain consisting of, or atleast comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ IDNo:10 and a VL domain consisting of, or at least comprising the VLCDR1,VLCDR2 and VLCDR3 aa sequences of, an aa sequence consisting of SEQ IDNo:32 (module P4/M57).

An anti-ErbB3 scFv antibody may comprise a VH domain comprising a set ofthree VHCDRs comprising VHCDR1, VHCDR2 and VHCDR3, and a VL domaincomprising a set of three VLCDRs comprising VLCDR1, VLCDR2 and VLCDR3,said CDRs comprising the aa sequences of SEQ ID NOs:309, 310 or 311,312, 313 or 314 (or 315), respectively, and wherein each CDR furthercomprises an amino terminus and a carboxy terminus, wherein the CDRs ofeach set of CDRs are arranged in the variable domain in a linear aminoto carboxy order of CDR1, CDR2 and CDR3, and wherein X aa in 309, 310 or311, 312, 313 or 314 (or 315) represent variable aa, which may be any aalocated in the corresponding position in FIG. 1 (for VH) and FIG. 2 (forVL). Anti-ErbB3 scFvs may comprise a VHCDR1, VHCDR2 and VHCDR3 of a VHdomain consisting of an aa sequence of the group of VH aa sequences inFIG. 3, e.g., consisting of SEQ ID NOs:134-165 and/or a VLCDR1, VLCDR2and VLCDR3 of a VL domain consisting of an aa sequence of the group ofVL aa sequences in FIG. 4, e.g., consisting of SEQ ID NOs:166-200. Incertain embodiments, an anti-ErbB3 scFv does not comprise all six CDRsof 16F or does not comprise either the VH domain of 16F and/or the VLdomain of 16F. For example an scFv comprises VH and VL aa sequences thatdiffer from those in 16F in at least one aa.

Anti-ErbB3 scFv antibodies may comprise a VH domain comprising the aasequence of SEQ ID NO:4 (or 5) and/or a VL domain comprising the aasequence set forth SEQ ID NO:6 (or 7), wherein the X aas in SEQ IDNOs:4, 5, 6 and 7 are variable aa which may be any aa at thecorresponding position in FIG. 3 (for the VH domain) and FIG. 4 (for theVL domain). Anti-ErbB3 scFv antibodies may comprise a VH domaincomprising an aa sequence in FIG. 3, e.g., selected from the groupconsisting of SEQ ID NOs:134-165, and/or a VL domain comprising an aasequence in FIG. 4, e.g., selected from the group consisting of SEQ IDNOs:166-200.

Exemplary anti-ErbB3 scFvs comprise a VH domain consisting of, or atleast comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of, an aasequence selected from the group of VH aa sequences consisting of SEQ IDNos:134-143 (the location of these CDRs is shown in FIG. 3). Anti-ErbB3scFvs may also comprise a VL domain consisting of, or at leastcomprising the VLCDR1, VLCDR2 and VLCDR3 aa sequences of, an aa sequenceselected from the group of VL aa sequences consisting of SEQ IDNos:166-175 (the location of these CDRs is shown in FIG. 4). In certainembodiments, anti-ErbB3 scFvs comprise a VH domain consisting of, or atleast comprising the VHCDR1, VHCDR2 and VHCDR3 aa sequences of, an aasequence selected from the group of VH aa sequences consisting of SEQ IDNos:134-143 and a VL domain consisting of, or at least comprising theVLCDR1, VLCDR2 and VLCDR3 aa sequences of, an aa sequence selected fromthe group of VL aa sequences consisting of SEQ ID Nos:166-175. Inparticular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:134 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:166 (module B60).In particular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:135 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:167 (B72). Inparticular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:136 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:168 (module M27).In particular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:137 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:169 (M7 module). Inparticular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:138 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:170 (P1 module). Inparticular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:139 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:171 (M27 module).In particular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:140 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:172 (B69 module).In particular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:141 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:173 (P6 module). Inparticular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:142 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:174 (M1.3 module).In particular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:143 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:175 (C8 module). Inparticular embodiments, an anti-ErbB3 scFv comprises a VH domaincomprising the VH domain consisting of, or at least comprising theVHCDR1, VHCDR2 and VHCDR3 aa sequences of, SEQ ID No:136 and a VL domainconsisting of, or at least comprising the VLCDR1, VLCDR2 and VLCDR3 aasequences of, an aa sequence consisting of SEQ ID No:169 (M27/M7module).

Exemplary scFvs are anti-IGF-1R scFv antibodies P4 (SEQ ID NO:367),M57(SEQ ID NO:368), M78, (SEQ ID NO:369), and M76 (SEQ ID NO:382); aswell as anti-ErbB3 scFv antibodies C8 (SEQ ID NO:370), P1 (SEQ IDNO:371), M1.3 (SEQ ID NO:372), M27 (SEQ ID NO:373), P6 (SEQ ID NO:374),B69 (SEQ ID NO:375) and P6L (SEQ ID NO:383).

scFvs may also comprise a CDR, a variable domain or their full length aathat differs from a CDR, variable domain, or full length scFv,respectively, set forth herein in one or more aa additions, deletions orsubstitutions, while retaining their binding properties. For example, aCDR may differ in 1 or 2 aa from a CDR sequence provided herein; avariable domain may differ in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aa from avariable domain sequence provided herein; and an scFv may differ in 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 aa from an scFv,respectively, provided herein. An scFv may also comprise a CDR, avariable domain or its full length aa sequence that is at least 70%,80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to a sequence of a CDR,variable domain or full length scFv sequence provided herein. In oneembodiment, an scFv comprises an aa sequence that is at least 70%, 80%,85%, 90%, 95%, 97%, 98%, or 99% identical to an aa sequence selectedfrom the group of scFv sequences consisting of SEQ ID NO:367, 368, 369,370, 372, 373, 374, and 375.

In certain embodiments, scFvs comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10aas at the amino terminus or carboxy terminus of the VL domain. Forexample, if the carboxy terminus of an scFv of interest would be an aathat can be clipped off by an enzyme, such as a carboxypeptidase (e.g.,a lysine or an arginine), one or more aa may be added to prevent the aafrom being clipped. For example, the aa “RT” from the CL domain may beadded to the carboxy terminus “VEIK” in anti-IGF-1R scFvs, as shown,e.g., in SEQ ID NOs:367-369.

Nucleic Acids, Expression Vectors and Host Cells

Provided herein are nucleic acids, e.g., DNA or RNA, encoding thepolypeptides described herein. Exemplary nucleotide sequences providedherein are those encoding the aa sequences of FIGS. 1-7, such as thenucleotide sequences of the Appendix, or portions thereof, such asportions that encode 1, 2, 3, 4 or 5 domains. Nucleotide sequences thatare at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to a nucleotide sequence encoding an aa sequence set forthherein, e.g., the nucleotide sequences set forth herein are alsoencompassed. Such nucleotide sequences may encode a protein set forthherein or may encode a protein that is at least 70%, 75%, 80%, 85%, 90%,95%, 97%, 98% or 99% identical or similar to a protein set forth hereinor a portion thereof (e.g., a domain), such as an aa sequence of any oneof FIGS. 1-7.

A nucleotide sequence encoding the heavy chain of 16F with a leadersequence (aa sequence SEQ ID NO:300) is set forth as SEQ ID NO:299. Anucleotide sequence encoding the light chain of 16F with a leadersequence (aa sequence SEQ ID NO:298) is set forth as SEQ ID NO:297.

In certain embodiments, a nucleic acid encodes the heavy and/or thelight chain of an antibody that comprises a leader sequence (or signalpeptide). An exemplary leader sequence is that shown in FIG. 7 for 16F.Accordingly, also provided herein are antibodies, e.g., those shown inFIGS. 5 and 6, linked to a leader sequence, such as that shown in FIG.7, and nucleic acids encoding such.

In certain embodiments, a nucleic acid is linked to a sequence thatenhances or promotes the expression of the nucleotide sequence in a cellto produce a protein. Such nucleic acids may be encompassed within avector, e.g., an expression vector. For expressing a protein that is atransmembrane protein, it is also preferable to include a signalsequence, e.g., the one of FIG. 7A, which signal sequence is frequentlydeleted to form a mature protein.

Also encompassed herein are cells, e.g., a host cell comprising anucleic acid or a vector provided herein.

The antibodies described herein may be produced by recombinant means.Methods for recombinant production are widely known in the state of theart and comprise protein expression in prokaryotic and eukaryotic cellswith subsequent isolation of the antibody and usually purification to apharmaceutically acceptable purity. For the expression of the antibodiesin a host cell, nucleic acids encoding the respective polypeptides,e.g., light and heavy chains, are inserted into expression vectors bystandard methods. Expression is performed in appropriate prokaryotic oreukaryotic host cells like CHO cells, NSO cells, SP2/0 cells, HEK293cells, COS cells, PER.C6 cells, yeast, or E. coli cells, and the bindingprotein is recovered from the cells (supernatant or cells after lysis).General methods for recombinant production of antibodies are well-knownin the art.

The antibodies may be suitably separated from the culture medium byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the antibodies are readily isolated and sequenced usingconventional procedures. The hybridoma cells can serve as a source ofsuch DNA and RNA. Once isolated, the DNA may be inserted into expressionvectors, which are then transfected into host cells such as HEK 293cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantantibodies in the host cells.

Aa sequence variants (e.g., mutants) of the antibodies may be preparedby introducing appropriate nucleotide changes into the antibody DNA, orby nucleotide synthesis.

“Host cell” denotes any kind of cellular system which can be engineeredto generate the antibodies described herein. In one embodiment, HEK293cells and CHO cells are used as host cells, in another CHO or NSO cellsare used.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

A nucleic acid is “operably linked” when it is placed in a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Purification of antibodies may be performed in order to eliminatecellular components or other contaminants, e.g., other cellular nucleicacids or proteins, by standard techniques, including alkaline/SDStreatment, CsCl banding, column chromatography, agarose gelelectrophoresis, and others well known in the art. Different methods arewell established and widespread used for protein purification, such asaffinity chromatography with microbial proteins (e.g., protein A orprotein G affinity chromatography), ion exchange chromatography (e.g.,cation exchange (carboxymethyl resins), anion exchange (amino ethylresins) and mixed-mode exchange), thiophilic adsorption (e.g., withbeta-mercaptoethanol and other SH ligands), hydrophobic interaction oraromatic adsorption chromatography (e.g., with phenyl-sepharose,aza-arenophilic resins, or m-aminophenylboronic acid), metal chelateaffinity chromatography (e.g., with Ni(II)— and Cu(II)-affinitymaterial), size exclusion chromatography, and electrophoretical methods(such as gel electrophoresis, capillary electrophoresis).

Methods of Using Antibodies Provided Herein

Provided herein are methods of using the antibodies described herein,e.g., an anti-IGF-1R+anti-ErbB3 PBA, an anti-IGF-1R antibody and ananti-ErbB3 antibody, for therapeutic applications. The antibodiesdisclosed herein can be used for treating a disease or disorderassociated with ErbB3 and/or IGF-1R dependent signaling, including avariety of cancers.

In one embodiment, a method is provided for inhibiting proliferation ofa tumor cell expressing IGF-1R and ErbB3 comprising contacting the tumorcell with an anti-IGF-1R+anti-ErbB3 bispecific (optionally polyvalent)antibody, such that proliferation of the tumor cell is inhibited, sloweddown, or stopped or such that the tumor cell dies.

Provided herein are methods for treating a disease or disorderassociated with ErbB3 and/or IGF-1R dependent signaling by administeringto a patient an antibody disclosed herein in an amount effective totreat the disease or disorder. Suitable diseases or disorders include,for example, a variety of cancers including, but not limited to, breastcancer and those set forth below. In one embodiment, a method fortreating a subject having a proliferative disease, such as cancer,comprises administering to a subject in need thereof a therapeuticallyeffective amount of one or more antibodies described herein, such as ananti-IGF-1R+anti-ErbB3 bispecific antibody.

Also provided is a method for (or a bispecific antibody e.g., in amedicament for) treating a tumor expressing IGF-1R and ErbB3 in apatient, the method comprising administering an effective amount of anantibody as described herein (e.g., effective to slow or stop tumorgrowth, or to shrink a tumor or to slow or stop tumor invasiveness ortumor metastasis). Any tumor expressing IGF-1R and ErbB3 may be treated,including tumors of the following cancers: lung cancer, sarcoma,colorectal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, head and neck squamous cell carcinoma (HNSCC), melanoma andbreast cancer. Particular examples of such tumors include: non-smallcell lung cancer, Ewing's sarcoma, tamoxifen-resistantestrogen-receptor-positive breast cancer, trastuzumab-resistant orlapatinib-resistant HER2-positive metastatic breast cancer,gefitinib-resistant or erlotinib-resistant lung cancer,cetuximab-resistant or panitumumab-resistant colorectal cancer,cetuximab-resistant head and neck squamous cell carcinoma (HNSCC), anderlotinib-resistant pancreatic cancer.

Also provided are kits comprising one or more antibodies disclosedherein. The kits may include a label indicating the intended use of thecontents of the kit and optionally including instructions for use of thekit in treating a disease or disorder associated with ErbB3 and/orIGF-1R dependent signaling, e.g., treating a tumor. The term labelincludes any writing, marketing materials or recorded material suppliedon or with the kit, or which otherwise accompanies the kit.

A method of treating a tumor herein provided can further compriseadministering a second anti-cancer agent in combination with theantibody. Thus novel pharmaceutical compositions are contemplatedcomprising an antibody disclosed herein, together with a secondanti-cancer agent, typically a biologic agent, together with at leastone pharmaceutically acceptable carrier or excipient.

Pharmaceutical Compositions

In another aspect, a composition, e.g., a pharmaceutical composition, isprovided for treatment of a tumor in a patient, as well as methods ofuse of each such composition to treat a tumor in a patient. Thecompositions provided herein contain one or more of the antibodiesdisclosed herein, formulated together with a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. Preferably, the carrier is suitablefor intravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the antibody may be coated in a material toprotect it from the action of acids and other natural conditions thatmay inactivate proteins.

Pharmaceutical compositions may be administered alone or in combinationtherapy, i.e., combined with other agents. For example, the combinationtherapy can include an antibody of the present disclosure with at leastone additional therapeutic agent, such as an anti-cancer agent.Pharmaceutical compositions can also be administered in conjunction withanother anti-cancer treatment modality, such as radiation therapy and/orsurgery.

A composition of the present disclosure can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

To administer a composition provided herein by certain routes ofadministration, it may be necessary or desirable to coat the antibodywith, or co-administer the antibody with, a material to prevent itsinactivation. For example, the antibody may be administered to a patientin an appropriate carrier, for example, in liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any excipient, diluent or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsprovided herein is contemplated. Supplementary active compounds (e.g.,additional anti-cancer agents) can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. Saline solutions and aqueousdextrose and glycerol solutions can be employed as liquid carriers,particularly for injectable solutions. The composition, if desired, canalso contain minor amounts of wetting or solubility enhancing agents,stabilizers, preservatives, or pH buffering agents. In many cases, itwill be useful to include isotonic agents, for example, sodium chloride,sugars, polyalcohols such as mannitol, sorbitol, glycerol, propyleneglycol, and liquid polyethylene glycol in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

EXAMPLES

The following examples should not be construed as limiting the scope ofthis disclosure.

Materials and Methods

Throughout the examples, the following materials and methods are usedunless otherwise stated. In general, the practice of the techniques ofthe present disclosure employs, unless otherwise indicated, conventionaltechniques of chemistry, molecular biology, recombinant DNA technology,immunology (especially, e.g., antibody technology), pharmacology,pharmacy, and standard techniques in polypeptide preparation.

Ligands

As used in these Examples and in the Figs., “HRG” refers to the isoformof heregulin known as heregulin 1 beta 1 (sometimes referred to asHRG1-B, HRG-β1, neuregulin 1, NRG1, neuregulin 1 beta 1, NRG1-b1, or HRGECD) e.g., R&D Systems, 377-HB-050/CF. As used in these Examples and inthe Figs., IGF-1 refers to insulin-like growth factor 1, e.g., R&DSystems, 291-GI-050/CF.

Cell Lines

All the human cell lines for use in the experiments described below maybe obtained, as indicated, from American Type Culture Collection (ATCC,Manassas, Va.) or the US National Cancer Institute (NCI) e.g., from theDivision of Cancer Treatment and Diagnostics (DCTD).

-   -   MCF7—ATCC® cat. No. HTB-22™    -   ADRr-NCI (redesignated NCI/ADR-RES)    -   BxPC-3—ATCC® cat. No. CRL-1687™    -   DU145—ATCC® cat. No. HTB-81™    -   Caki-1—ATCC® cat. No. HTB-46™    -   SK-ES-1—ATCC® cat. No. HTB-86™

The mouse anti-human-IGF-1R monoclonal antibody mAb391 (IgG1, R & DSystems MAB391) is used as an anti-IGF-1R IgG antibody control.

Example 1 Rational Engineering of Antibody Therapeutics TargetingMultiple Signaling Pathways

The ErbB pathway has long been the focus of cancer research due to thehigh expression of the ErbB receptors in specific cancer types:HER2/ErbB2 is gene amplified in some breast cancers, and EGFR/ErbB1 ishighly expressed in colon cancers and NSCLC. For two members of thepathway EGFR/ErbB1 and HER2/ErbB2, there are approved monoclonalantibody agents (e.g., cetuximab and trastuzumab) and small moleculetyrosine kinase inhibitors (e.g., erlotinib, lapatinib). Thesetherapeutics perturb the ability of extracellular stimuli to activatedownstream intracellular signaling networks; however, it is difficult todetermine what represents an optimal therapeutic strategy given that theErbB signaling network is quite complex (FIG. 9A). In addition toEGFR/ErbB1 and HER2/ErbB2 (devoid of ligand binding activity), there aretwo other members of ErbB pathway—ErbB3 (kinase-dead) and ErbB4. Allfour receptors can dimerize with each other to various degrees followingligand activation, contributing to an essential step required forintracellular signal transmission. Following dimerization, the receptorscan be internalized and recycled at rates that depend on the type ofdimer, as well as on the activation status of downstream signalingpathways, such as the PI3K pathway.

The complexity of the ErbB network, combined with the ability to measurethe abundance and activation state of key components, lends the networkto computational modeling. By using computational modeling a number ofbiological phenomena can be well described, such as thedimerization-dependence of receptor trafficking, control of signalamplification through feedback loops and ligand-dependence of signalpropagation. For example, to determine an optimal strategy to inhibitthe ErbB pathway, a network model was built to describe the activationof the PI3K/AKT pathway in response to the ligands betacellulin andheregulin, which selectively activate EGFR/ErbB1 or ErbB3 heterodimers,respectively. The mechanistic model represented the processes of: ligandbinding; receptor dimerization; receptor trafficking and signalpropagation, with a series of reactions defined by mass-action kinetics.In order to make reliable predictions, mechanistic models have to befirst trained using temporal and dose-dependent experiments that capturekey dynamic events, specifically the activation of the ErbB receptorsand PI3K/AKT pathway. Network components with the greatest influencewere identified by sensitivity analysis, where each component of thenetwork is subtly perturbed and the relative contribution to thedownstream output is assessed (FIG. 9B).

Using these methods a computational model of the ErbB network wasgenerated, which identified the kinase-dead ErbB3 as the strongestactivator of the PI3K/AKT pathway. In fact, despite its low expressionlevel, in the presence of either heregulin or betacellulin ErbB3provided the strongest contribution toward activation of the PI3K/AKTpathway in the model. Notably, this in silico observation applied evento cell lines expressing relatively low levels of ErbB3 and 10-foldhigher levels EGFR/ErbB1 or HER2/ErbB2.

In addition to identifying optimal targets, mechanistic modeling can bealso used to determine optimal therapeutic design characteristics. Inthe case of targeting the kinase-dead ErbB3, optimization simulations ofa therapeutic monoclonal antibody explored several designcharacteristics, such as binding to ErbB3, preventing heregulin binding,and blockade of dimerization, with a special focus on blockingligand-induced EGFR/ErbB3 dimerization. Simulation was used to determinethe affinity required to achieve maximal inhibition of AKTphosphorylation through simulation of inhibitors within a range ofdissociation rate constants. From this simulation, a 1 nanomolaraffinity was predicted to be sufficient for maximal inhibitor potency,with higher affinity inhibitors displaying only limited improvement(FIG. 9C and FIG. 9D).

The added complexity of a bispecific agent interacting with a biologicalsystem creates an even greater opportunity to utilize mechanisticmodeling to guide engineering efforts. All bispecific proteins bindtheir targets in a manner dependent on the affinity for each target,avidity-enhanced crosslinking ability and the relative abundance of eachtarget. Designing a bispecific optimized for potent inhibition alsorequires knowledge of the affinity of competing ligands and dimerizationpartners, if such exist, as well as the relative strength of each targetin activating common downstream signaling cascades and subsequent cellgrowth and survival mechanisms. The desirable apparent Kd can beachieved through multiple rounds of affinity and avidity improvements,and the simulation model can guide engineering efforts towards the mostsuitable molecular format and streamlined optimization route. Simulationcan be used to explore the performance of a bispecific protein conceptfor permutations of target affinity, avidity and target expressionlevels.A Bispecific Antibody Designed to Inhibit Two Cell Surface Growth FactorReceptors (IGF-1R, ERBB3) with a Single Binding Moiety Directed TowardsEach Target

All bispecific proteins bind their targets in a manner dependent on theaffinity for each target, avidity-enhanced crosslinking ability and therelative abundance of each target; the added complexity of a bispecificinteracting with a biological system creates an even greater opportunityto utilize mechanistic modeling to guide engineering efforts. Designinga bispecific optimized for potent inhibition also requires knowledge ofthe affinity of competing ligands and dimerization partners, if suchexist, as well as the relative strength of each target in activatingcommon downstream signaling cascades and subsequent cell growth andsurvival mechanisms. The desirable apparent Kd can be achieved throughmultiple rounds of affinity and avidity improvements, and the simulationmodel can guide engineering efforts towards the most suitable molecularformat and streamlined optimization route. Simulation can be used toexplore the performance of a bispecific protein concept for permutationsof target affinity, avidity and target expression levels.

Simulating the dose-response behavior of a bispecific antibody designedto inhibit two cell surface growth factor receptors (IGF-1R and ErbB3)with a single binding moiety directed towards each target in this systemreveals that IGF-1R is more potently inhibited when ErbB3 is more highlyexpressed and less potently inhibited when ErbB3 more scarcely expressed(FIG. 10A). Therefore, the ability of the bispecific antibody to inhibitIGF-1R depends on its avid binding. This phenomenon is specific to boththe relative expression of the targets and the relative affinities ofthe bispecific antibody towards the targets: inhibition of ErbB3 is lessaffected by the expression of IGF-1R as the bispecific antibody issimulated to bind to ErbB3 with a higher affinity than IGF-1R (FIG.10B). For a bispecific inhibitor this receptor level-dependent behaviorcan seriously limit overall efficacy, as depicted in the simulatedeffect on a downstream intracellular readout common to both pathways,AKT: poor inhibition of pAKT is predicted when ErbB3 is under-expressedas IGF-1R is not sufficiently inhibited (FIG. 10C). Simulation of thismodel system reveals that the performance of bispecific inhibitorstowards each target can be highly dependent on the relative expressionof both targets; information that can be useful for therapeutic design.

The impact of receptor level dependence on the potency of a bispecificcan be extensively explored through simulation of many hypotheticalcancer cells where the expression level of each target is varied over aclinically-relevant range and the IC50 of each target and downstreamreadout is calculated and plotted on a response surface. The shape ofthe IC50 response surface depends on the underlying pathwayinteractions. ErbB3 is the stronger activator of downstream signaling,and defines the area where the bispecific inhibitor would be mostefficacious. Simulation of a bispecific with single binding moieties toeach target reveals that the most potent inhibition of the downstreamtarget is centered on regions of equal target expression. In fact, wheneither target is overexpressed by as little as 5-fold the IC50 value canincrease as much as 100-fold, with a substantial loss in potency.

Mapping actual target levels in tumor cell lines or clinical samplesonto the IC50 response surface can be used to guide therapeuticimprovement efforts by revealing if the region of most potent inhibitionoverlaps with the relevant patient population. Strategies to shift theregion of predicted optimal potency to treat a different or broaderpatient population can be explored first through simulation. IgG-likebispecific antibodies have two binding moieties towards each target andtherefore exhibit same-target avidity in addition to cross-targetavidity, improving the effective binding affinity for each target.Simulation of the IgG-like bispecific antibody that has monovalentbinding affinities equal to the bivalent bispecific protein shows thatthis format is less dependent on cross-target avidity for performance:The region of optimal potency is broader than the monovalent bispecific.Therefore, if the goal is to treat a broad patient population the modelprediction would be to use an IgG-like bispecific design.

The benefit of affinity maturation of antibody function in improving theregion of optimal potency can also be examined through simulation.Simulating downstream target inhibition across the receptor space by anon-optimal tetravalent bispecific identifies areas of poor inhibition,particularly when IGF-1R is more highly expressed. Increasing themonovalent binding affinity of the bispecific towards IGF-1R by 10-foldthrough lowering the dissociation rate predicts the improvement affinitymaturation would achieve. The downstream target is inhibitedsignificantly more potently both where the IGF-1R is more highlyexpressed and where the ErbB3 is more highly expressed indicating thataffinity maturation towards one target is enhancing the potency towardsthe second target through cross-target avidity.

These considerations were set as criteria for an optimization campaignof a proof-of-concept IgG-like bispecific therapeutic protein thatshowed potent inhibition of growth-factor induced signaling and tumorinhibition in xenograft models validating the design criteria. Thisprotein comprised an IgG antibody framework directed at IGF-1R and twoC-terminally fused scFv modules directed at ErbB3, but was not suitablefor downstream development, as it contained unstabilized scFv modulesthat do not have sufficient intrinsic stability. This phenomenon is wellunderstood and engineering of scFv modules for stability has beendescribed using a variety of techniques, including linker optimization,disulfide engineering, targeted mutagenesis, co-variation analysis, loopgrafting on stable framework, structure-guided design, focused designand phage display.

To combine optimization of the scFv modules affinity and stabilitywithin a single campaign a combination of structure-guided design, yeastsurface display and micro scale biophysical characterization was used.Structure-guided scFv variants were designed where stability enhancingmutations were introduced, motifs conferring potential CMC liabilitieswere mutated, atypical framework aas were removed or replaced, andvariation in low diversity portions of the CDRs was introduced. Sinceyeast cells have eukaryotic posttranslational modification andpolypeptide export machinery, surface expression levels that werereported to predict thermal stability and soluble secretion efficiencywere monitored. In addition a thermal challenge “cook-and-bind” protocolwas developed. In this experiment the unstable scFv modules unfolded,while the stable high affinity proteins retained binding to the antigenand therefore were enriched (FIG. 11A). After isolation of individualclones, scFvs fused to the yeast surface were challenged and clones wereselected based on the residual affinity measured by a flow cytometry(FIG. 11B). Thermostable scFv modules that showed over 10-foldimprovement in Kd on the yeast surface were produced as soluble proteinsand those showing improved antigen binding, inhibition of growth factorsignaling, and acceptable stability were selected. These optimized scFvmodules were C-terminally fused to the IGF-R1 antibody. The resultingIgG-like proteins were expressed in a transient expression system,purified using protein A chromatography, and profiled using biophysical,biochemical, and cell signaling assays. Among many useful biophysicaltechniques, differential scanning fluorescence and thermal inactivationassays were found to be most informative at the 1 to 5 microgram scale.Micro-scale triage composed of differential scanning fluorescenceprofiling and thermal inactivation assays allow the selection ofbispecifics with improved serum and aggregation stabilities.Differential scanning fluorescence profiling and thermal inactivationassays give complementary information on the rate of unfolding and therate of aggregation for the least stable protein domain. These dataqualitatively predict the serum and aggregation stability of an IgG-likebispecific antibody (FIG. 12). The importance of having a robustsensitive micro scale assay is difficult to overestimate, as it directlytranslates into the ability to interrogate a larger number of diversecandidates within a single design campaign. The improved potency andstability of the engineered IgG-like bispecific protein was confirmedusing a normal scale assay: binding to BxPC-3 cells that express bothtargets (FIG. 13). This demonstrates that an approach featuring parallelfocused engineering of the modules of a multifunctional protein,followed by high-throughput production and characterization is generallyapplicable to improving potency and manufacturability of targetedbispecific antibody-like proteins in the context of one therapeuticdesign cycle.

Example 2 Preparation, Expression and Purification of IgG TetravalentBispecific Proteins

Three anti-ErbB3-anti-IGF-1R IgG2 tetravalent bispecific proteins(“ELI-7,” “ILE-10” and “ILE-12”) and other control proteins for use inthe experiments described in Example 3, were prepared essentially asfollows. ELI-7, ILE-10 and ILE-12 have the structure IgG2 (scFv)₂.

ELI-7 is an anti-ErbB3/anti-IGF-1R IgG2 tetravalent bispecific proteincomprising an anti-ErbB3 IgG2 antibody, to which an anti-IGF-1R scFv islinked to each of the C-termini of the heavy chains of the IgG2 protein.

ILE-10 and ILE-12 are anti-IGF-1R/anti-ErbB3 IgG2 tetravalent bispecificproteins comprising an anti-IGF-1R IgG2 antibody, to which an anti-ErbB3scFv is linked to each of the C-termini of the heavy chains of the IgG2protein.

The structure and relationships of ELI-7, ILE-10 and ILE-12 are setforth in Table 6. Briefly, they all comprise the same anti-IGF-1R VHsequence (“module 5-7”). ILE-10 and ILE-12 differ only in the sequenceof the ErbB3 scFv. ILE-10 and ELI-7 comprise the same anti-IGF-1R andanti-ErbB3 VH sequences, and differ in that ILE-10 has an IGF-1R Fab andan ErbB3 scFv (“ILE”) and ELI-7 has the opposite configuration (“ELI”).The control antibodies are monospecific and each comprises a bindingsite homologous to ones found in the bispecific antibodies.

TABLE 6 Description of proteins used in Examples 2 and 3 Anti-IGF-1RAnti-ErbB3 module module Orientation ELI-7 5-7 2-3 ErbB3 - IGF-1R ILE-105-7 2-3 IGF-1R - ErbB3 ILE-12 5-7  2-21 IGF-1R - ErbB3 Anti-IGF-1R Ab5-7 — — module 5-7 Anti-ErbB3 Ab — 2-3 — module 2-3 Anti-ErbB3 Ab — 2-21 — module 2-21

Much of the disclosures of Examples 2-3 herein, including ELI-7, ILE-10and ILE-12, are found in PCT application PCT/US2010/052712.

The aa sequences of the light and heavy chains of each of the proteinsare as follows: Heavy chain of ELI-7: SEQ ID NO:316. Light chain ofELI-7: SEQ ID NO:317. Heavy chain of ILE-10: SEQ ID NO:318. Heavy chainof ILE-12: SEQ ID NO:319. Light chain of ILE-10 and IL-12: SEQ IDNO:320.

The nucleic acids encoding the proteins (referred to as “fusionproteins”) are cloned as single proteins into the expression plasmidsusing standard recombinant DNA techniques. An expression vector employedis pMP 10K (SELEXIS). Expression plasmids are linearized, purified usingQIAquick® purification kit (QIAGEN), and co-transfected into CHO cellsusing Lipofectamine™ LTX (Invitrogen). Transfected cells are recoveredwith F12Hams medium containing 10% FBS for 2 days without selectionpressure, then with selection pressure for 4 days. After 4 days, theyare changed into serum-free medium (Hyclone) containing glutamine withselection pressure. After a week, cells are checked for expression andscaled up to desired volume. All proteins are purified using acombination of three chromatography steps: protein A affinity, cationexchange and anion exchange. Each is carried out in accordance with themanufacturer's instructions. The protein A affinity step is used toselectively and efficiently bind the fusion proteins out of harvestedcell culture fluids (HCCF). This removes >95% of product impurities in asingle step with high yields and high throughput. The portion of desiredmolecular form for fusion proteins after this step was in the range of60 to 98 percent. MABSELECT from GE is used as the Protein A affinityresin. SPFF (sulphopropyl fast flow) from GE, an agarose based resin, isused as the cation exchange resin in the second chromatography step. Theportion of desired molecular form for fusion proteins after this stepwas in the range of 90 to 99 percent. QSFF (Quaternary-amine sepharosefast flow) from GE, an agarose based anion exchange resin, is used in athird and final chromatography step. The purified material wasconcentrated and dialyzed into PBS. The final yield for the fusionproteins after this step was is in the range of 20 mg-100 mg/L.

Example 3 Binding and Biological Activity of Anti-ErbB3+Anti-IGF-1R IgGTetravalent Bispecific Proteins

This Example shows that an anti-IGF-1R+anti-ErbB3 IgG tetravalentbispecific protein (ELI-7) binds with high affinity to IGF-1R and toErbB3 (as also shown for two similar proteins ILE-10 and ILE-12),potently inhibits 1) signal transduction from the IGF-1R and ErbB3receptors, 2) AKT phosphorylation, and 3) tumor cell proliferation invitro and in vivo. Results were obtained essentially as follows.

A) Binding of ELI-7, ILE-10 and ILE-12 to IGF-1R and ErbB3

1×10⁵ MCF7 cells or 1×10⁵ ADRr cells are incubated at room temperaturefor 2 hours with each of ELI-7, ILE-10 and ILE-12, two anti-ErB3antibodies and one anti-IGF-1R antibody at 2 uM, followed by 12subsequent 3-fold dilutions. Then using goat anti-HSA-Alexa647conjugated antibody as the detection antibody, cells are incubated onice for 40 minutes. Cell binding dissociation constants (measures ofbinding affinities) of the antibodies on MCF7 and ADRr cells areassessed by FACS and apparent dissociation constants are determined foreach protein. The following results were obtained (see also FIGS. 14Aand 14B):

TABLE 7 Binding Kds of bispecific proteins Kd (nM) Inhibitor ADRr (n =3) MCF7 (n = 1) ELI-7 2.5 2.1 ILE-10 7.1 4.5 ILE-12 0.3 0.6 Anti-ErbB3IgG 0.4 0.04 (module 2-21) Anti-ErbB3 Ig 1.2 0.9 (module 2-3)Anti-IGF-1R Ig 5.1 5.6 (module 5-7)

The results show that IgG-bispecifics (i.e. ELI-7, ILE-10, ILE-12) boundto both cell types, in some cases with greater binding at lowconcentrations, indicating avid binding and the ability to bind to eachreceptor. The IgG-bispecifics had a similar Kd to the equivalentmonoclonal antibody component.

B) Signal Inhibition of IGF-1R, ErbB3 and Akt by ELI-7 and ILE-7

The ability of ELI-7 and ILE-7 to antagonize IGF-1R and ErbB3 andinhibit activation (phosphorylation) of downstream components, IGF-1R,ErbB3 and Akt; phosphorylation is examined. 3.5×10⁴ BxPC-3 cells arepre-incubated for 1 hour with an antibody at 0.3 μM, followed by 9subsequent 3-fold dilutions to give a 10-point curve. Cells are treatedwith IGF-1 at 80 ng/ml and heregulin at 20 ng/ml for 15 minutes.Phosphorylation of IGF-1R to yield phospho-IGF-1R (pIGF-1R) is measuredby ELISA (R & D Systems; Cat.# DYC1770) to evaluate the ability of theagents to inhibit pIGF-1R formation. Phosphorylation of ErbB3 ismeasured by ELISA (R & D Systems; Cat.#DYC1769) to evaluate the abilityof the agents to inhibit pErbB3 formation. Phosphorylation of AKT ismeasured by ELISA using the following antibodies: anti-AKT, clone SKB1(Millipore, Cat.#05-591); biotinylated anti-phospho-AKT(Ser⁴⁷³-specific; Cell Signaling Technology Cat.#5102). ILE-7 is atrivalent protein having the same binding sites as ELI-7 and describedin PCT/US2010/052712. FIGS. 15A-15C show the results that were obtainedessentially as described above for ILE-7 and ELI-7. The results thatwere obtained are also summarized in the table below.

TABLE 8 Inhibition of phosphorylation of ErbB3, IGF-1R and AKT by ELI-7Ki (nM) ELI-7 ILE-7 pAKT 6.3 1.3 pErbB3 1.3 0.6 pIGF-1R 14.1 0.8

The results show that ELI-7 inhibits phosphorylation of ErbB3, IGF-1Rand Akt, even with simultaneous stimulation with IGF-1 and HRG.

C) Cell Growth Inhibition by ELI-7 in Two Dimensional Culture

The effect of ELI-7 on tumor cell proliferation is examined in vitrousing a CTG assay, which is a luminescence-based assay that measures theamount of cellular ATP present (Promega; Cat.# PR-G7572), indicated asRelative Light Units (RLU). 500 cells per well of DU145 cells areincubated for 6 days in medium with 80 ng/ml IGF-1 and 20 ng/ml HRG andcontaining a 3-fold dilution of inhibitors starting at 2 uM. The controlconsists of DU145 cells incubated without growth factors or antibodies.

Results obtained essentially as described above indicate that the ELI-7inhibited the growth of DU145 cells (Ki=12 nM, see FIG. 16), whereasinhibitors of either IGF-1R or ErbB3 had no effect on cell growth.

A similar experiment was conducted on another cell line. 2000 BxPC-3cells per well are incubated for 6 days in medium containing a 3-folddilution of inhibitors starting at 1 uM. The control consists of IgG2Kappa from human myeloma plasma” (Sigma Aldrich catalog #I5405).

Results obtained essentially as described above indicate that ELI-7inhibited BxPC-3 growth by 46% (p<0.001, Student's T-test) (FIG. 17).

D) Tumor Growth Inhibition by Bispecific Proteins in Human XenograftMouse Models of Cancer

This example shows that ELI-7 inhibits tumor growth in mouse models ofcancer in two different models.

First the pharmacokinetic properties of each bispecific protein in micewas calculated. 600 ug of ELI-7 or 500 ug of each HSA-linked trivalentcontrol protein (ILE-3, ILE-7, and ILE-9; described inPCT/US2010/052712) was injected via tail vein into each mouse (4 miceper inhibitor and time point). Blood was drawn at various time pointsthereafter (mice were first sacrificed and then blood was drawn bycardiac puncture). Time points for ELI-7 are: 0.5, 4, 24, 72, 120, 168and 240 hours. Time points for trivalent control proteins are: 0.5, 4,8, 24, 28, 72, and 120 hours. For ELI-7, blood concentration is measuredusing an anti-human IgG ELISA kit (Bethyl labs Cat.# E80-104) accordingto the manufacturer's instructions. For the trivalent proteins,concentration in the blood is measured using an ELISA kit that detectsIGF-1R and ErbB3 binding specifically, plates are coated with His-taggedhuman IGF-1R, incubated with the trivalent proteins or ELI-7, thendetected with a human ErbB3-Fc (R&D Systems) and an anti-Fc-HRPdetection reagent. Pharmacokinetic properties (half-life and Cmax) foreach protein are calculated using a one-compartment model. The followingresults were obtained:

TABLE 9 Half-life and Cmax of ELI-7 in mouse blood Half life CmaxAntibody (hours) (ug/ml) ELI-7 48 612 ILE-3 15 410 ILE-7 14 516 ILE-9 17447 anti-IGF-1R IgG 124 517 (module 5-7) anti-ErbB3 IgG 58 645 (module2-3)

Simulation of drug-specific half-lives led to prediction that thefollowing doses would result in equal exposure (or in the case of ILE-750% comparable exposure):

TABLE 10 Predicted dose for equal exposure Table 10 Dose (ug) ELI-7 600ILE-7 800 anti-IGF-1R IgG 300 (module 5-7) anti-ErbB3 IgG 500 (module2-3)

The effect of ELI-7 on human pancreatic cancer xenograft tumor growth inmouse models was then assessed by injecting 5×10⁶ BxPC-3 cells(resuspended in a 1:1 mixture of PBS and growth factor-reduced matrigel;BD Biosciences Cat.#354230) into the subcutaneous space in the flank ofeach mouse. Tumors were allowed to develop for 7-10 days (until theyreached a volume of approximately 100-200 mm³), and then tumor size wasmeasured for each mouse (pi/6×length×width², where width is the smallestmeasurement). Mice were then size-matched and then randomly assignedinto treatment groups. ELI-7, a trivalent control protein (ILE-7), ananti-ErbB3 antibody, an anti-IGF-1R antibody or a PBS control was theninjected every 3 days until the completion of the study.

The results, (FIGS. 18 and 19), show that ELI-7 significantly inhibitedthe xenograft tumor growth of BxPC-3 tumors compared to the PBS control:final tumor volume was 77% lower in ELI-7 treated tumors compared to thePBS control (p values determined by student's T-test). Day 0 refers tothe first day of dosing.

The effect of ELI-7 on human prostate cancer xenograft tumor growth in amouse model was assessed by injecting 5×10⁶ DU145 cells (resuspended ina 1:1 mixture of PBS and growth factor-reduced matrigel; BD BiosciencesCat.#354230) into the subcutaneous space in the flank of each mouse.Tumors were allowed to develop for 7-10 days (until each reached avolume of approximately 100-200 mm³), and then tumor size was measuredfor each mouse (pi/6×length×width², where width is the smallestmeasurement). Mice were then tumor-size-matched and then randomlyassigned into the treatment groups. ELI-7, a trivalent control protein(ILE-7), an anti-ErbB3 antibody, an anti-IGF-1R antibody or a PBScontrol was then injected every 3 days until the completion of thestudy.

The results, (FIGS. 20A and 20B), show that ELI-7 significantlyinhibited xenograft tumor growth of DU145 cells, whereas the controlanti-IGF-1R and anti-ErbB3 antibodies did not: the final tumor volumewas 50% lower in ELI-7 treated tumors compared to the PBS control (pvalues determined by student's T-test). Day 0 refers to the first day ofdosing.

E) ELI-7 Inhibits Signaling Across a Broad Range of ErbB3 and IGF-1RReceptor Levels

To determine whether ELI-7 can inhibit downstream signaling across abroad range of ErbB3 and IGF-1R receptor levels the following experimentwas performed:

BxPC-3 cell receptor levels are varied by shRNA-mediated knockdown ofIGF-1R or ErbB3 in BxPC-3 cells using the pLKO.1 PURO vector (Sigma).The shRNA sequences are provided in PCT/US2010/052712. ErbB3 and IGF-1Rlevels are then measured by quantitative FACS and the mean receptorlevels are calculated from the resulting distribution (see Table 11 forrelative expression levels). To determine the potency of ELI-7, cellsare serum-starved and pretreated with ELI-7 for 1 hour at 37° C.,followed by a 15-minute stimulation with 20 ng/ml HRG+80 ng/ml IGF1.Signal inhibition is assessed by ELISA for pAKT.

The results indicate that ELI-7 displayed similar potency across theBxPC-3 cells lines with modified receptor levels as indicated by theirIC50 values and overlapping confidence intervals (see Table 11),indicating that ELI-7 has broad activity against a range of receptorprofiles (FIG. 21).

TABLE 11 Relative receptor levels and pAkt IC50 values for four BxPC-3cell lines: Engineered % of control pAkt 95% Confidence Sigma-AldrichBxPC-3 cell line receptor level IC50 Interval Catalog # BxPC-3-non-IGF-1R and ErbB3 3.6 nM 0.9-14.7 nM SHC002V targeted control levelsunchanged BxPC-3-IGF-1R- IGF-1R level 6.4 nM 2.9-14.1 nM SHCLNV- mod.1reduced by 37% NM_000875- TRCN0000039673 BxPC-3-ErbB3- ErbB3 level 3.3nM 1.4-8.0 nM SHCLNV- mod.1 reduced by 48% NM_001982- TRCN0000230091BxPC-3-ErbB3- ErbB3 level 7.6 nM 1.2-50.0 nM SHCLNV- mod.2 reduced by88% NM_001982- TRCN0000018327

Example 4 Biological Activity of an Anti-IGF-1R/Anti-ErbB3 TetravalentBispecific Protein with Enhanced Activities (16F) Relative to ELI-7

The proof of concept protein (ELI-7) described in Examples 2 and 3 wasfurther improved to increase its binding affinity to IGF-1R and ErbB3,biological activity, stability and solubility; as described inExample 1. The following changes were made: (i) the orientation wasswitched from anti-ErbB3 as the IgG component to anti-IGF-1R as the IgGcomponent; (ii) an anti-ErbB3 binding moiety binding to a differentepitope was used; (iii) its CDR3 VH region was affinity matured; (iv)the anti-IGF-1R IgG component was mutated to stabilize it (stabilizingmutations) and (v) its backbone was switched from IgG2 to IgG1. Theresulting protein is 16F, whose aa sequences are of FIG. 7.

The increase in anti-IGF-1R potency of 16F relative to that of ELI-7, asa function of inhibition of IGF-1R phosphorylation, is measured asdescribed in Example 3. The results that were obtained essentially asdescribed above, (FIG. 22), indicate that the reengineered protein is asignificantly more potent inhibitor of IGF-1R signal transduction.

The potency of inhibition of signal transduction through ErbB3 andthrough inhibition of AKT phosphorylation is measured essentially asdescribed in Example 3 for 16F, ELI-7 and a combination of ANTI-IGF-1RAb# A (ganitumab; SEQ ID 327+SEQ ID 328) and anti-ErbB3 Ab# A (SEQ ID336+SEQ ID 337). These measurements are carried out in BxPC-3 cells inthe presence of HRG and IGF1. The results that were obtained essentiallyas described above, (Table 12), indicate that 16F has improved efficacyin inhibiting signal transduction compared to ELI-7, which efficacy iscomparable to that of a combination of the clinical grade inhibitorsANTI-IGF-1R Ab# A+anti-ErbB3 Ab# A.

TABLE 12 Comparison of 16F with Eli-7 and clinical-grade inhibitorspErbB3 pIGF-1R pAKT IC50 IC50 IC50 Inhibitor (nM) (nM) (nM) ELI-7 3.7 109.4 16F 0.5 1.1 2.5 ANTI-IGF-1R Ab# A 0.8 0.9 2.2 (ganitumab; SEQ ID NO:327 + SEQ ID NO: 328) + ANTI-ErbB3 Ab# A (SEQ ID NO:336 + SEQ ID NO:337)

As described in Example 1, it was also shown that the re-engineeredbispecific, i.e., 16F, is more thermal and serum stable than ELI-7. Inaddition, 16F is less prone to aggregation: (i) 16F is stable at 19mg/ml in PBS at 4° C., with only about 2% aggregation in 33 days; (ii)no significant change in % monomers was observed after 3 freeze thawcycles; (iii) no significant change in % monomers was observed aftershaking at 4° C. for one day; and (iv) no significant change in %monomers was observed on incubation at 37° C. for 6 days.

Results from additional comparative experiments described below alsoshow that 16F is at least as effective as a combination of commercialanti-IGF-1R and anti-ErbB3 in inhibiting signal transduction.

In a first set of experiments, the effectiveness of 16F (SF-G1-C8) ininhibiting the phosphorylation IGF-1R, ErbB3 or AKT was compared to thatof the Anti-IGF-1R Ab#B (cixutumumab; SEQ ID 324+SEQ ID 325), Anti-ErbB3Ab# A (SEQ ID 336+SEQ ID 337) or Anti-IGF-1R Ab#B+Anti-ErbB3 Ab# A intwo different cell lines (BxPC-3 and DU145).

BxPC-3 and DU145 cells are maintained in RPMI-1640 medium supplementedwith 10% fetal bovine serum, Penicillin/Streptomycin and L-glutamine.For signaling experiments, 3.5×10⁴ cells are plated in complete mediumin 96-well tissue culture plates. The following day, complete medium isreplaced with serum-free medium, and cells are incubated overnight at37° C. Cells are pretreated for 1 hour with the indicated doses ofantibody, and then stimulated for 15 minutes with 100 ng/ml IGF-1(Calbiochem) and 30 ng/ml HRG (R&D Systems). Cells are washed with PBSand lysed in MPer buffer supplemented with protease and phosphataseinhibitors.

ELISAs for phospho-IGF-1R (pIGF-1R) and phospho-ErbB3 (pErbB3) arepreformed according to the manufacturer's protocols (R&D Systems). AnELISA for phospho-AKT (pAKT) is performed with the following reagents:anti-AKT capture antibody (Millipore), anti-pAKT (Ser473) detectionantibody (Cell Signaling), and streptavidin-HRP(R&D Systems).SUPERSIGNAL ELISA PICO chemiluminescent substrate (Pierce) is added andplates read on a PerkinElmer EnVision® plate reader. Luminescence valuesare plotted and IC50 values calculated using Graphpad Prism 5 software.

The results, which were obtained essentially as described above and areshown in FIG. 23 (BxPC-3 cells) and FIG. 24 (DU145 cells) and in Table13, indicate that 16F shows surprisingly more potent inhibition of dualpathway signaling through pErbB3 and pAKT than the combination ofanti-IGF-1R Ab# B and anti-ErbB3 Ab# A.

TABLE 13 IC50 values for inhibitor treatments presented in FIG. 23. CellpIGF-1R pErbB3 pAKT Line Inhibitor IC50 IC50 IC50 BxPC-3 16F (SF-G1-C8)7.7E−10 2.4E−10 2.3E−09 BxPC-3 Anti-IGF-1R Ab# B 2.4E−09 ND 1.4E−08(cixutumumab; SEQ ID NO: 324 + SEQ ID NO: 325) BxPC-3 ANTI-ErbB3 Ab# AND 5.1E−11 3.9E−10 (SEQ ID NO: 336 + SEQ ID NO: 337) BxPC-3 ANTI-IGF-1RAb# B + 1.9E−09 3.2E−10 3.2E−09 ANTI-ErbB3 Ab# A DU145 16F (SF-G1-C8)1.1E−09 2.0E−10 5.1E−10 DU145 Anti-IGF-1R Ab# B 9.1E−10 ND 9.8E−09(cixutumumab; SEQ ID NO: 324 + SEQ ID NO: 325) DU145 ANTI-ErbB3 Ab# A ND8.2E−11 2.8E−10 (SEQ ID NO: 336 + SEQ ID NO: 337) DU145 ANTI-IGF-1R Ab#B + 1.0E−09 3.2E−10 9.3E−10 ANTI-ErbB3 Ab# A

In a second set of experiments, the effectiveness of 16F (SF-G1-C8) ininhibiting the phosphorylation IGF-1R, ErbB3 or AKT was compared to thatof the anti-IGF-1R antibody ANTI-IGF-1R Ab# A, the anti-ErB3 antibodyanti-ErbB3 Ab# A, or a combination of the latter two antibodies inBxPC-3 cells.

The results, which were obtained essentially as described above and areshown in FIG. 24 and Table 14, indicate surprisingly more potentinhibition of dual pathway signaling through pErbB3 and pIGF-1R by 16Fthan by the combination of ANTI-IGF-1R Ab# A and anti-ErbB3 Ab# A.

TABLE 14 IC50 values for inhibitor treatments presented in FIG. 24.pErbB3 pIGF-1R pAKT Inhibitor IC50 IC50 IC50 16F (SF-G1-C8) 5.0E−101.1E−09 2.5E−09 ANTI-IGF-1R Ab# A ND 1.5E−10 2.6E−10 (ganitumab; SEQ IDNO: 327 + SEQ ID NO: 328) ANTI-ErbB3 Ab# A 4.3E−10 ND 5.7E−09 (SEQ IDNO: 336 + SEQ ID NO: 337) ANTI-IGF-1R Ab# A + 8.2E−10 9.3E−10 2.2E−09ANTI-ErbB3 Ab# A

Example 5 Binding and Biological Activity of AdditionalAnti-IGF-1R/Anti-ErbB3 IgG Tetravalent Bispecific Antibodies Comprisingthe SF Module

Additional anti-IGF-1R+anti-ErbB3 IgG tetravalent bispecific antibodieswere constructed. Each of these PBAs was assembled by combining threemodules, essentially as shown in FIG. 8. Each PBA comprises a pair ofheavy chain fusion polypeptides (each comprising at least a part of eachof the three modules), each member of the heavy chain pair being boundto the other and each further being bound to one of a pair of lightchains. The three modules assembled into each PBA are:

-   -   1. an N-terminal (amino terminal) Fab variable domain module        comprising both (essentially identical) light chains and the        N-termini of both heavy chains;    -   2. an scFv module; and    -   3. a HC IgG CR module interposed between the N-terminal Fab        variable domain module and the scFv module.

The heavy chains being fusion polypeptides comprise the heavy chainportion of the N-terminal Fab module, an IgG CR module and theC-terminal scFv module.

The new anti-IGF-1R+anti-ErbB3 antibodies were made of a combination ofthe anti-IGF-1R and anti-ErbB3 moieties of Table 15, assembled asmodules arranged in differing orientations. For each of Tables 15 and16, each PBA that was built comprises a fusion protein comprising a pairof essentially identical heavy chain polypeptides, each comprising acombination, in N-terminal to C-terminal (amino to carboxy) order, ofeach Fab module named in the left column with an IgG1 CR (G1) and withany one scFv module named in the right column of the same Table. The aasequences of the heavy and light chains of these additional PBAs are ofFIG. 5A (anti-IGF-1R+anti-ErbB3) and FIG. 5B (anti-ErbB3 andanti-IGF-1R).

TABLE 15 Anti-IGF-1R-anti-ErbB3 proteins Anti-IGF-1R Anti-ErbB3 Fab scFvSF C8 P4 P1 M78 M1.3 M57 M27 P6 B69

TABLE 16 Anti-ErbB3-anti-IGF-1R proteins Anti-ErbB3 Anti-IGF-1R Fab scFvP1 P4 M27 M78 M7 B72 B60

This example shows that the antibodies that comprise an N-terminal “SF”module bind BxPC-3 cells, bind ErbB3, inhibit IGF-1R, ErbB3 and AKTphosphorylation, and are stable. Results obtained with the otherproteins are of Example 6.

A) BxPC-3 Cell Binding Data

Binding of SF-G1-P1, SF-G1-P6, SF-G1-M27, SF-G1-B69, SF-G1-M1.3 andSF-G1-C8 (16F) to BxPC-3 was measured essentially as follows.

BxPC-3 cells are maintained in RPMI-1640 medium supplemented with 10%fetal bovine serum, Penicillin/Streptomycin and L-glutamine. Medium wasremoved and the BxPC-3 cells were washed with PBS. Trypsin is addeduntil the cells detached from the plate, and then neutralized withmedium+10% serum. The cells are spun down and resuspended in FACS buffer(1×PBS+2% Serum+0.1% Azide). Aggregates are broken down into singlecells by pipetting up and down and putting the cells through a cellstrainer. The cells are spun down and resuspended in FACS buffer at adensity of 2×10⁶ cells/ml. In a 96 well conical bottom plate, 50 ul ofcell suspension is aliquotted per well to give 10⁵ cells/well.

Antibodies are diluted to 1 uM in FACS buffer, and 10 3-fold dilutionsare done, with a final well consisting of FACS buffer only (no primaryantibody). 50 ul of antibodies are added to 50 ul of cells so that thehighest final antibody concentration is 500 nM in the first well. Cellsand antibodies are incubated at room temperature with gentle agitationfor 2 hrs. The plates are spun at 1,500 RPM for 5 min, and thesupernatant is removed. Pellets are washed three times in FACS buffer.After the final wash the FACS buffer is removed, and 50 ul ofant-Fc-DyLight 649 secondary antibody (Abcam) added at 1:100 in FACSbuffer. Cells are incubated in the cold room in the dark with gentleagitation for 1 hour, washed again three times and resuspended in 100 ulfixing buffer (PBS with 1% Paraformaldehyde, 2% FBS). The samples aretransferred to 96 well U-bottom FACS plates (Becton Dickinson) and keptin the dark at 4 degrees until use. Samples were read using aFACSCalibur (Becton Dickinson), and Median Fluorescent Intensities (MFI)were determined using FlowJo. The analysis was performed with GraphPadPRISM, using a log (agonist) vs response (three parameter) non-linearregression curve fit.

The results (FIG. 25) and in Table 17, indicate that these bispecificantibodies display strong binding to BxPC-3 cells.

TABLE 17 EC50 values for bispecific antibody binding presented in FIG.25. Bispecific Antibody EC50 (nM) SF-G1-C8 3.1 SF-G1-P1 4.9 SF-G1-P6 2.9SF-G1-M27 2.7 SF-G1-B69 2.1 SF-G1-M1.3 3.5

B) ErbB3 Binding Data

Binding of SF-G1-P1, SF-G1-P6, SF-G1-M27, SF-G1-B69, SF-G1-M1.3 andSF-G1-C8 (16F) to recombinant ErbB3 was measured essentially as follows.

96-well REACTI-BIND plates (Pierce) are coated with 50 ul of ErbB3-His(ErbB3 with a C-terminal hexa-histidine tag—2 ug/ml in PBS)(“hexa-histidine” disclosed as SEQ ID NO:403) and incubated overnight at4° C. The next day plates are washed with PBS+0.05% Tween-20 (PBS-T) andblocked for 1 hr. at room temperature with 100 ul of Protein-FreeBlocking Buffer (Pierce). Plates are washed with PBS-T and 50 ul of eachbispecific antibody is added in duplicate. Concentrations start at 500nM (in PBS-T) and include ten additional two-fold dilutions and oneblank (PBS-T only). Plates are incubated at room temperature for twohours and then washed with PBS-T. 50 ul of anti-Fc-HRP (Jackson Labs) isadded at 1:40,000 in PBS-T, and plates are incubated in the dark for 1hr. at room temperature. Plates are again washed with PBS-T and 100 ulof TMB substrate (Thermo Scientific, TMB and peroxide solution mixed1:1) added. The plates are incubated for 5-15 minutes at roomtemperature until a blue color develops, and the reaction is stoppedwith 100 ul of STOP solution (Cell Signaling Technology). The absorbancewas read at 450 nm on a PerkinElmer Envision plate reader, and bindingcurves were generated with GraphPad PRISM, using a log (agonist) vsresponse (three parameter) non-linear regression curve fit.

The results (FIG. 26 and Table 18), indicate that the bispecificantibodies display strong binding to recombinant ErbB3 protein.

TABLE 18 EC50 values for bispecific antibody binding presented in FIG.26. Bispecific Antibody EC50 (nM) SF-G1-C8 0.3 SF-G1-P1 0.4 SF-G1-P6 0.3SF-G1-M27 0.4 SF-G1-B69 0.4 SF-G1-M1.3 0.2

C) Inhibition of Signal Transduction

Inhibition of signal transduction by SF-G1-P1, SF-G1-P6, SF-G1-M27,SF-G1-B69, SF-G1-M1.3 and SF-G1-C8 (16F) was measured essentially asfollows.

BxPC-3 cells are maintained in RPMI-1640 media supplemented with 10%fetal bovine serum, Penicillin/Streptomycin and L-glutamine. 3.5×10⁴cells are plated in complete medium in 96-well tissue culture plates.The following day, complete medium is replaced with serum-free medium,and cells incubated overnight at 37° C. Cells are pretreated for 1 hourwith the indicated doses of drug, and then stimulated for 15 minuteswith 100 ng/ml IGF1 (Calbiochem) and 30 ng/ml HRG (R&D Systems). Cellsare washed with PBS and lysed in MPer buffer (“Mammalian ProteinExtraction Reagent” Pierce Thermo Scientific) supplemented with proteaseand phosphatase inhibitors.

ELISAs for phospho-IGF1R (pIGF1R) phospho-ErbB3 (pErbB3) and phospho-AKT(pAKT) are preformed as described in Example 4, above. Relativeluminescence units (RLU) were plotted and IC₅₀ values calculated usingGraphpad Prism 5 software.

The results (FIG. 27 and Table 19), indicate that the bispecificproteins strongly inhibit dual pathway signaling.

TABLE 19 IC50 values and percent inhibition values for inhibitortreatments shown in FIG. 27. pIGF1R pIGF1R Inhibitor IC50 % InhibitionSF-G1-P6 8.2E−10 91.2 SF-G1-M1.3 8.0E−10 87.9 SF-G1-B69 1.2E−09 91.1SF-G1-P1 9.2E−10 91.3 SF-G1-M27 6.0E−10 90.7 SF-G1-C8 9.5E−10 93.0pErbB3 pErbB3 IC50 % Inhibition SF-G1-P6 2.9E−10 96.6 SF-G1-M1.3 2.5E−1097.0 SF-G1-B69 5.2E−10 97.8 SF-G1-P1 6.9E−10 95.3 SF-G1-M27 2.5E−10 98.1SF-G1-C8 2.4E−10 94.9 pAKT pAKT IC50 % Inhibition SF-G1-P6 1.9E−09 75.6SF-G1-M1.3 1.2E−09 77.4 SF-G1-B69 2.7E−09 72.7 SF-G1-P1 2.4E−09 71.7SF-G1-M27 1.4E−09 73.8 SF-G1-C8 1.5E−09 72.3

D) Stability of the Bispecific Proteins

Various stability studies have been performed and they show thatSF-G1-P1, SF-G1-P6, SF-G1-M27, SF-G1-B69, SF-G1-M1.3 are stable inserum, are thermally stable, and are stable at low pH.

For determining serum stability, the proteins are incubated in mouseserum (Sigma) at a final concentration of 2.5 uM for either 0 hr or 72hrs at 37° C. The samples are then assayed using the colorimetric ELISAbinding assay described above, and binding curves are generated withGraphPad Prism. Absorbance values are normalized to 0 hr at theinflection point of each curve to determine the percent binding retainedafter 72 hrs in serum at 37° C.

Results that were obtained essentially as described above, (FIG. 28),indicate that each of the PBAs tested has a (normalized) serum stabilityafter 72 hours of at least 70%. Certain PBAs have a stability of about100%.

For determining thermal stability, EC90 values were calculated for eachPBA using the binding curves generated in the ELISA binding experimentdescribed above. Each PBA is prepared at 5× its EC90 value in PBS andtransferred to PCR plates (Bio-Rad) at 50 ul per well. The plates arespun down and placed in the ICYCLER IQ gradient PCR machine (Biorad) toheat the antibodies for 1 hr from 47-72° C. Aliquots of each antibodyare also kept at 25° C. and 37° C. for 1 hr. The plates are then spundown at 2,000 RPM for 5 minutes, and supernatants are diluted five-foldin PBS-T to their EC90 concentration. The samples are then assayed usingthe colorimetric ELISA binding assay described above, and absorbancesare normalized to 25° C. Binding curves were generated with GraphPadPrism to determine T₅₀ values.

Results that were obtained essentially as described above, (Table 20),indicate that the T₅₀ values vary from 46.7° C. to 62.6° C.

TABLE 20 T₅₀ values for each bispecific antibody incubated at 25-72° C.for 1 hr Bispecific Antibody T₅₀ SF-G1-C8 62.1° C. SF-G1-P1 46.7° C.SF-G1-P6 62.4° C. SF-G1-M27   56° C. SF-G1-B69 62.6° C. SF-G1-M1.3 46.7°C.

The temperature at which the PBAs unfold was determined by DifferentialScanning Fluorimetry (DSF). The DSF assay is performed in the IQ5 RealTime Detection System (Bio-Rad). 20 μl solutions of 15 uM bispecificantibody, 1× Sypro Orange (Invitrogen Life Technologies), and 1×PBS wereadded to the wells of a 96 well plate. The plate was heated from 20° C.to 90° C. with a heating rate of 1° C./min. Data was transferred toGraphPad Prism for analysis.

Results that were obtained essentially as described above, (Table 21),indicate that the proteins unfold at different temperatures.

TABLE 21 T_(m) values for each bispecific antibody, as determined by DSFBispecific Antibody T_(m) SF-G1-C8 69° C. SF-G1-P1 54° C. SF-G1-P6 61°C. SF-G1-M27 55° C. SF-G1-B69 61° C. SF-G1-M1.3 64° C.

For determination of pH 3 stability, SF-G1-C8 stock solution is dilutedinto 0.1 M acetic acid (pH 3.0) and incubated for 1 hour. The solutionis then neutralized with 1M Tris Base, dialyzed against PBS andconcentrated. The dialysate is tested by SEC (Size ExclusionChromatography) and colorimetric ELISA against a sample of SF-G1-C8neutralized immediately after protein A purification. SEC is performedusing Agilent 1100 Series HPLC system. 50 ug of SF-G1-C8 is injected ona TSK Super SW3000 gel column (Tosoh Biosciences, P/N 18675). PBS isused as running and equilibration buffer at a flow rate of 0.35 ml/min.The ELISA is performed as described above, coating the plates witheither recombinant IGF1R-His or ErbB3-His.

Results that were obtained essentially as described above indicate thatSF-G1-C8 is stable, with binding to IGF1R and ErbB3-His substantiallyunaffected, after low pH incubation (pH 3) for 1 hour.

For determining the stability of SF-G1-C8 for an extended time at 4° C.,SF-G1-C8 (19 mg/ml) was incubated in PBS at 4° C. for either 1, 6 or 33days and subjected to SEC. Percent monomer was determined by SEC asdescribed above. The results indicate that SF-G1-C8 displays 98%stability after 33 days at 4° C.

Example 6 Characterization of Additional Anti-IGF-1R/ErbB3 andAnti-ErbB3/IGF-1R PBAs A) Binding to BxPC-3 Cells

Binding of PBAs to BxPC-3 cells is determined as follows. BxPC-3 cellsare maintained in RPMI-1640 media supplemented with 10% fetal bovineserum, Penicillin/Streptomycin and L-glutamine. Medium is removed andthe BxPC-3 cells are washed with PBS. Trypsin is added until the cellsdetached from the plate, and is then neutralized with media+10% serum.The cells are spun down and resuspended in FACS buffer (1×PBS+2%Serum+0.1% azide). Aggregates are broken down into single cells bypipetting up and down and putting the cells through a cell strainer. Thecells are spun down and resuspended in FACS buffer at a density of 1×10⁶cells/ml. In a 96 well conical bottom plate, 50 ul of cell suspension isaliquotted per well to give 5×10⁴ cells/well.

PBAs are diluted to 2 uM in FACS buffer, and 10 3-fold dilutions aredone, with a final well consisting of FACS buffer only (no primaryantibody). 50 ul of the serially diluted antibodies are added to 50 ulof cells so that the highest final antibody concentration is 1 uM in thefirst well. Cells and antibodies are incubated at room temperature withgentle agitation for 2 hrs. The plates are spun at 1,500 RPM for 5 min,and the supernatant removed. Pellets are washed three times in FACSbuffer. After the final wash the FACS buffer is removed, and 50 ul ofanti-Fc-DyLight 649 secondary antibody (Abcam) is added at 1:100 in FACSbuffer. Cells are incubated in the cold room in the dark with gentleagitation for 1 hour, washed again three times, and resuspended in 100ul fixing buffer (PBS with 1% Paraformaldehyde, 2% FBS). The samples aretransferred to 96 well U-bottom FACS plates (Becton Dickinson) and keptin the dark at 4 degrees until use. Samples were read using a FACSCalibur (Becton Dickinson), and Median Fluorescent Intensities (MFI)were determined using FlowJo. One Site—Total Binding was used todetermine EC50 values with GraphPad PRISM.

Results that were obtained essentially as described above and are shownin FIG. 29 (A-C) and in Table 22 below, indicate that the PBAs displaystrong binding to BxPC-3 cells. FIG. 29(D) and Table 22 below displaythe binding data analyzed using a One Site—Total Binding curve fit.

TABLE 22 EC50 values from the separate binding experiments presented ineach of FIGS. 29A-D Bispecific Antibody EC50 (nM) FIG. 29A SF-G1-C8(16F) 0.6 M27-G1-P4 1.2 M27-G1-M57 1 M27-G1-M78 2.1 B60-G1-P4 0.5B60-G1-M57 0.3 B60-G1-M78 0.3 M27/M7-G1-P4 2.2 M27/M7-G1-M57 2M27/M7-G1-M78 1.7 FIG. 29B SF-G1-C8 (16F) 0.3 M57-G1-M1.3 0.2 M57-G1-P60.2 M57-G1-V8 0.1 M78-G1-M1.3 0.2 M78-G1-P6 0.4 M78-G1-V8 0.3 FIG. 29CSF-G1-C8 (16F) 0.3 P4-G1-M1.3 0.03 P4-G1-P6 0.02 P4-G1-V8 0.1 M7-G1-M570.6 M7-G1-M78 1 M7-G1-P4 2.8 FIG. 29D SF-G1-C8 1.2 SF-G1-P1 2 SF-G1-P61.1 SF-G1-M27 1.5 SF-G1-B69 1.1 SF-G1-M1.3 1.4

B) Inhibition of Cell Signaling

Inhibition of cell signaling by PBAs is determined essentially asfollows. BxPC-3 cells are maintained in RPMI-1640 media supplementedwith 10% fetal bovine serum, Penicillin/Streptomycin and L-glutamine.3.5×10⁴ cells are plated in complete medium in 96-well tissue cultureplates. The following day, complete medium is replaced with serum-freemedium, and cells are incubated overnight at 37° C. Cells are pretreatedfor 1 hour with the indicated doses of drug, and then stimulated for 15minutes with 100 ng/ml IGF1 (Calbiochem) and 30 ng/ml HRG (R&D Systems).Cells are washed with PBS and lysed in MPer buffer supplemented withprotease and phosphatase inhibitors.

ELISAs for phospho-IGF1R (pIGF1R) phospho-ErbB3 (pErbB3) and phospho-AKT(pAKT) are performed as described in Example 4, above.

ILE-10 (14F or 14f)) and ELI-7 (5F or 5f), both of which are describedabove, are used as reference proteins.

Results that were obtained essentially as described above and are shownin FIGS. 30 (pIGF-1R), 31 (pErbB3) and 32 (pAKT), and in Table 23 below,indicate that the PBAs display strong inhibition of dual pathwaysignaling.

TABLE 23 IC50 values and percent inhibition values for inhibitortreatments shown in FIGS. 30, 31 and 32 IC50 % Inhibition IC50 %Inhibition IC50 % Inhibition pIGF1R pIGF1R pErbB3 pErbB3 pAKT pAKT ErbB3IgG-IGF1R scFv B60-G1-P4 4.4E−09 88 4.0E−11 99 5.5E−10 86 B60-G1-M571.0E−08 85 1.2E−09 162 7.3E−10 83 B60-G1-M78 3.5E−09 86 8.0E−11 1156.4E−10 78 M27-G1-P4 3.0E−09 88 1.0E−10 106 5.7E−10 90 M27-G1-M578.1E−09 83 3.1E−10 88 1.3E−09 84 M27-G1-M78 1.4E−09 87 1.2E−10 1076.6E−10 92 M27/M7-G1-P4 1.3E−08 99 4.0E−11 111 2.5E−10 92 M27/M7-G1-M577.5E−09 91 1.5E−10 179 5.3E−10 91 M27/M7-G1-M78 1.0E−09 91 1.1E−10 1403.5E−10 92 M7-G1-M57 2.10E−08  82 5.2E−11 124 2.8E−10 88 M7-G1-M781.30E−09  88 2.6E−11 111 3.7E−10 95 M7-G1-P4 2.40E−09  93 2.4E−11 1091.8E−10 95 IGF1R IgG-ErbB3 scFv M57-G1-M1.3 4.1E−10 94 1.4E−10 992.3E−09 85 M57-G1-P6 2.5E−10 92 7.9E−11 98 2.1E−09 79 M57-G1-C8 2.5E−1093 6.5E−11 98 2.0E−09 80 M78-G1-M1.3 5.0E−10 97 1.4E−10 99 2.2E−09 92M78-G1-P6 4.2E−10 95 1.4E−10 100 2.1E−09 86 M78-G1-C8 6.8E−10 96 2.3E−1098 4.1E−09 84 P4-G1-M1.3 1.8E−10 92 5.5E−11 94 1.1E−09 86 P4-G1-P61.6E−10 91 3.9E−11 95 1.2E−09 82 P4-G1-C8 1.3E−10 91 3.6E−11 94 1.2E−0978 SF-G1-C8 5.2E−10 91 1.9E−10 98 3.9E−09 71

In another set of experiments, the level of inhibition of ligand-inducedsignal transduction by the PBAs was compared to that obtained with priorart anti-IGF-1R (anti-ErbB3 Ab# A—SEQ ID NO:336 for HC and 337 for LC)and prior art anti-ErbB3 (ANTI-IGF-1R Ab# A—SEQ ID NO:327 for the HC andSEQ ID NO:328 for the LC) antibodies. The experiments were conductedessentially as described immediately above in this Example.

The results, (FIGS. 33, 34 and 35 and Table 24), indicate that the PBAsshow surprisingly high levels of inhibition of dual pathway signalingrelative to the combination of ANTI-IGF-1R Ab# A and anti-ErbB3 Ab# A.

TABLE 24 IC50 values for inhibitor treatments presented in FIGS. 33, 34and 35 Cell pIGF1R pErbB3 pAKT Line Inhibitor IC50 IC50 IC50 BxPC-3B60-G1-M57 4.4E−09 2.3E−11 6.5E−10 BxPC-3 B60-G1-M78 1.4E−09 1.0E−113.4E−10 BxPC-3 B60-G1-P4 2.2E−08 ~2.4e−14    1.1E−10 BxPC-3 M7-G1-M572.1E−08 5.2E−11 2.8E−10 BxPC-3 M7-G1-M78 1.3E−09 2.6E−11 3.7E−10 BxPC-3M7-G1-P4 2.4E−09 2.4E−11 1.8E−10 BxPC-3 P4-G1-M1.3 1.1E−09 5.3E−113.4E−10 BxPC-3 P4-G1-C8 1.3E−09 5.8E−07 3.1E−10 BxPC-3 SF-G1-C8 2.6E−092.1E−10 6.5E−10 BxPC-3 ANTI-IGF-1R Ab# A 1.6E−09 4.3E−09 8.0E-11(ganitumab; SEQ ID 327 + SEQ ID 328) BxPC-3 anti-ErbB3 Ab# A 2.3E−109.2E−11 3.5E-10 (SEQ ID 336 + SEQ ID 337) BxPC-3 ANTI-IGF-1R Ab# A +1.5E−09 9.4E−11 4.3E-10 anti-ErbB3 Ab# A

C) Stability of the Bispecific Proteins

Various stability studies were performed essentially as described inExample 5D above, and their results show that the PBAs tested werestable in serum and were thermally stable.

Serum stability results, (FIG. 36), indicate that the PBAs display somedifferences in serum stability. The lowest stability was about 65% andthe hightest stability was about 100%. Those that have a number of about1 (or above) are considered to have about 100% stability.

Melting temperatures results are of Table 25. The results indicate thatthe PBAs unfold at varying temperatures.

TABLE 25 T_(m) values for each bispecific antibody, as determined byDSF. M27/M7 refers to a binding site having an M27 heavy chain and an M7light chain. Bispecific Antibody Tm (° C.) B60-G1-P4 66.5 B60-G1-M5767.8 B60-G1-M78 67.5 M27-G1-P4 66.5 M27-G1-M57 67.2 M27-G1-M78 66.8M27/M7-G1-P4 66.5 M27/M7-G1-M57 68.5 M27/M7-G1-M78 67.5 M57-G1-M1.3 64.5M57-G1-P6 65.5 M57-G1-C8 66.5 M78-G1-M1.3 68.5 M78-G1-P6 66.5 M78-G1-C8— P4-G1-M1.3 62.5 P4-G1-P6 63.5 P4-G1-C8 66.5 M7-G1-M57 70.5 M7-G1-M7867.5 M7-G1-P4 66.5SEC stability results are shown in Table 26, and indicate that the PBAsare mostly monomeric.

TABLE 26 Percent monomer determined by SEC for each bispecific antibodyBispecific Antibody Percent Monomer B60-G1-P4 91 B60-G1-M57 92B60-G1-M78 87 M27-G1-P4 84 M27-G1-M57 87 M27-G1-M78 83 M27/M7-G1-P4 90M27/M7-G1-M57 93 M27/M7-G1-M78 78 M7-G1-P4 82.4 M7-G1-M57 87.4 M7-G1-M7882 M57-G1-M1.3 79 M57-G1-P6 77 M57-G1-C8 77 M78-G1-M1.3 79 M78-G1-P6 80M78-G1-C8 77 P4-G1-M1.3 92 P4-G1-P6 86 P4-G1-C8 95

Example 8 Identification of Additional High Affinity Anti-IGF-1R andAnti-ErbB3 Binding Domains

Many more high affinity anti-IGF-1R and anti-ErbB3 binding domains wereisolated via phage screening. The sequences of the heavy and light chainof these proteins are of FIGS. 1-4 under the 16F sequences. Thesequences of anti-IGF-1R binding sites start with “5-7” and thesequences of anti-ErbB3 binding sites start with “E3B.”

Example 9 Potent Inhibition of Dual IGF1- and HRG-Stimulated Signalingby Anti-IGF1R-Anti+ErbB3 BPAs

This Example shows that anti-IGF1R+anti-ErbB3PBAs are potent inhibitorsof dual IGF1 and HRG-stimulated signal transduction in DU145 and BxPC-3cells.

Results were obtained essentially as follows. 35,000 BxPC-3 cells areplated in 10% serum overnight at 37° C. The following day, cells arestarved in media containing 0.5% serum and incubated overnight at 37° C.Cells were pretreated for one hour with the indicated concentrations ofAb, and then stimulated for 15 minutes with 30 ng/ml HRG1b1-ECD+100ng/ml IGF1. In this Example, and in Examples 10-13 and 21, controlanti-IGF1R and anti-ErbB3 mAb are ANTI-IGF-1R Ab# A and anti-ErbB3 Ab#A, respectively. Cells are lysed in M-Per buffer (+protease/phosphataseinhibitors) and run on ELISA for pAKT. For the pAKT ELISA assay, platesare coated with anti-AKT (Millipore), blocked with PBS+2% BSA, incubatedwith lysates and standards, and detected with a biotinylated anti-pAKT(Ser473) and streptavidin-HRP. ELISA pico chemiluminescent substrate isadded and plates are read on a Perkin-Elmer Envision plate reader. AllIC50 curves and calculated values are generated in Graphpad Prism.

The results, (FIG. 39), indicate that PBAs M7-G1-M78, P4-G1-M1.3,P4-G1-C8 and SF-G1-C8 potently inhibit signal transduction induced byIGF-1 and HRG in both DU145 and BxPC-3 cells, as determined by measuringphosphorylated AKT (pAKT).

Example 10 Anti-IGF1R+Anti-ErbB3 BPA Potency is Maintained Over a BroadRange of Receptor Profiles

This Example shows that anti-IGF1R+anti-ErbB3 PBAs are potent inhibitorsof dual IGF1 and HRG-stimulated signal transduction in cells havingvarious levels of IGF1R or ErbB3.

Results were obtained essentially as follows. BxPC-3 cells are infectedwith lentivirus expressing a control hairpin, or with shRNA specific toIGF1R or ErbB3 (Sigma) that reduces expression of these proteins byabout 50%. Knockdown is confirmed by FACS and Western blot analysis.Cells are plated and treated as described in Example 9. Levels of pAKTare determined as described in Example 9.

The results, (FIG. 40 and Table 27), indicate that PBAs M7-G1-M78,P4-G1-M1.3, P4-G1-C8 and SF-G1-C8 potently inhibit signal transductioninduced by IGF-1 and HRG in BxPC-3 cells having high or lower levels(50% reduced levels) of IGF1R or ErbB3, as determined by measuringphosphorylated AKT (pAKT).

TABLE 27 PBAs IC50 (in M) and percent inhibition of results shown inFIG. 40 pAKT pAKT Cell Line Inhibitor % Inhibition IC50 BxPC-3 (VectorControl) M7-M78 89.8 1.1E−09 BxPC-3 (Vector Control) P4-M1.3 87.97.1E−10 BxPC-3 (Vector Control) P4-C8 78.3 1.4E−09 BxPC-3 (VectorControl) SF-C8 68.8 3.5E−09 BxPC-3 (Vector Control) ANTI-IGF-1R Ab# A38.2 2.1E−08 (Ganitumab; SEQ ID 327 + SEQ ID 328) BxPC-3 (VectorControl) ANTI-ErbB3 Ab# A 47.2 2.5E−09 (SEQ ID 336 + SEQ ID 337) BxPC-3(Vector Control) ANTI-IGF-1R Ab# A + 90.6 3.3E−09 ANTI-ErbB3 Ab# ABxPC-3 (50% IGF1R KD) M7-M78 90.9 3.4E−10 BxPC-3 (50% IGF1R KD) P4-M1.391.1 3.8E−10 BxPC-3 (50% IGF1R KD) P4-C8 80.4 7.6E−10 BxPC-3 (50% IGF1RKD) SF-C8 74.5 6.3E−09 BxPC-3 (50% IGF1R KD) ANTI-IGF-1R Ab# A 514.0E−08 (Ganitumab; SEQ ID 327 + SEQ ID 328) BxPC-3 (50% IGF1R KD)ANTI-ErbB3 Ab# A 58.2 1.6E−09 (SEQ ID 336 + SEQ ID 337) BxPC-3 (50%IGF1R KD) ANTI-IGF-1R Ab# A + 89.8 1.3E−09 ANTI-ErbB3 Ab# A BxPC-3 (50%ErbB3 KD) M7-M78 91.2 4.0E−10 BxPC-3 (50% ErbB3 KD) P4-M1.3 90.7 3.2E−10BxPC-3 (50% ErbB3 KD) P4-C8 90.9 3.3E−10 BxPC-3 (50% ErbB3 KD) SF-C882.7 2.1E−09 BxPC-3 (50% ErbB3 KD) ANTI-IGF-1R Ab# A 47.7 1.3E−07(Ganitumab; SEQ ID 327 + SEQ ID 328) BxPC-3 (50% ErbB3 KD) ANTI-ErbB3Ab# A 59.7 2.8E−10 (SEQ ID 336 + SEQ ID 337) BxPC-3 (50% ErbB3 KD)ANTI-IGF-1R Ab# A + 92.8 6.0E−10 ANTI-ErbB3 Ab# A BPAs are indicatedwith “G1” in Table 27. For example, “M7-M78” refers to “M7-G1-M78. ”

Example 11 Anti-IGF1R+Anti-ErbB3 PBAs are Potent Inhibitors of Low Doseor High Dose IGF1- or HRG-Induced Signaling

This Example shows that anti-IGF1R+anti-ErbB3 PBAs are potent inhibitorsof dual IGF1 and HRG-stimulated signal transduction in response to highor low ligand (IGF1 or HRG) concentration.

The results were obtained essentially as follows. 35,000 BxPC-3 cellsare plated in 10% serum overnight at 37° C. The following day, cells arestarved in media containing 0.5% serum and incubated overnight at 37° C.Cells are pretreated for one hour with the indicated concentrations ofPBA, and then stimulated for 15 minutes with either low (40 ng/ml) orhigh (400 ng/ml) IGF1, or low (20 ng/ml) or high (200 ng/ml) HRG1b1-ECD.ELISA for pErbB3, pIGF1R, and tIGF1R are from commercial sources (R & DSystems). For the pAKT ELISA assay, plates are coated with anti-AKT(Millipore), blocked with PBS+2% BSA, incubated with lysates andstandards, and detected with a biotinylated anti-pAKT (Ser473) andstreptavidin-HRP. ELISA pico chemiluminescent substrate is added andplates are read on a Perkin-Elmer Envision plate reader. All IC50 curvesand calculated values are generated in Graphpad Prism.

The results, (FIGS. 41, 42 and Table 28), indicate that the PBAsM7-G1-M78, P4-G1-M1.3, P4-G1-C8 and SF-G1-C8 potently inhibit signaltransduction induced by higher or lower levels of IGF-1 and HRG inBxPC-3 cells, as determined by measuring pAKT (pAKT), pIGF-1R and pErbB3levels.

TABLE 28 PBA IC50s (in M) and percent inhibition of results shown inFIGS. 41 and 42 Ligand Ligand Conc. Readout M7-G1-M78 P4-G1-C8P4-G1-M1.3 SF-G1-C8 ANTI-IGF-1R Ab# A (ganitumab; SEQ ID NO: 327 + SEQID NO: 328) IGF1 40 ng/ml pIGF1R 7.80E−10 1.00E−09 1.10E−10 1.20E−105.10E−10 IGF1 400 ng/ml pIGF1R 2.50E−10 2.40E−09 8.80E−11 9.90E−114.40E−10 IGF1 40 ng/ml pAKT 7.40E−09 3.80E−10 3.80E−10 1.40E−10 1.30E−09IGF1 400 ng/ml pAKT 9.10E−09 6.40E−10 3.40E−10 2.10E−10 5.90E−10ANTI-ErbB3 Ab# A (SEQ ID NO: 336 + SEQ ID NO: 337) HRG 20 ng/ml pErbB32.41E−10 6.16E−11 6.76E−11 3.12E−11 2.69E−10 HRG 200 ng/ml pErbB31.98E−10 6.53E−11 5.70E−11 2.72E−11 2.66E−10 HRG 20 ng/ml pAKT 3.06E−107.12E−11 1.06E−10 3.97E−11 4.32E−10 HRG 200 ng/ml pAKT 3.26E−10 6.46E−111.01E−10 4.09E−11 5.50E−10

Example 12 Anti-IGF1R+Anti-ErbB3 PBAs Suppress Basal Signaling

This Example shows that anti-IGF1R+anti-ErbB3 PBAs inhibit basal levelsof signal transduction.

Results were obtained essentially as follows. 35,000 BxPC-3 cells areplated in 10% serum overnight at 37° C. The following day, cells arestarved in media containing 0.5% serum and incubated overnight at 37° C.Cells are pretreated for either 15 minutes or 24 hours in the presenceof the indicated concentration of Ab, but in the absence of ligandstimulation. Cells are lysed in M-Per buffer (+protease/phosphataseinhibitors) and run on ELISA for pAKT, as described in Example 9.

The results, (FIG. 43), indicate that PBAs M7-G1-M78, P4-G1-M1.3, andP4-G1-C8 suppress the basal level of pAKT.

Example 13 Anti-IGF1R+Anti-ErbB3 PBAs Potently Downregulate IGF1R

This Example shows that anti-IGF1R+anti-ErbB3 PBAs downregulate IGF1R.

Results were obtained essentially as follows. 35,000 BxPC-3 cells areplated in 10% serum overnight at 37° C. The following day, cells arestarved in media containing 0.5% serum and incubated overnight at 37° C.Cells are then incubated for 24 hours in media containing 0.5% serum andthe indicated concentrations of antibody (starting at a high dose of5E-07M with subsequent 3-fold dilutions). Cells are lysed and totalIGF1R is measured by ELISA using a commercial kit from R&D Systems.Percent downregulation is calculated from Prism using the followingformula: 100*((fit.max−min.observed)/(fit.max−no stimulation).

The results, (FIG. 44 and Table 29), indicate that PBAs M7-G1-M78,P4-G1-M1.3, and P4-G1-C8 reduce the level of IGF1R in A549 and BxPC-3cells.

TABLE 29 Potent Downregulation of IGF1R by PBAs. % IGF1R Cell LineMolecule Downregulation A549 M7-G1-M78 48 A549 P4-G1-C8 70 A549P4-G1-M1.3 72 A549 ANTI-IGF-1R Ab# A 57 (Ganitumab; SEQ ID 327 + SEQ ID328) BxPC-3 M7-G1-M78 53 BxPC-3 P4-G1-C8 67 BxPC-3 P4-G1-M1.3 71 BxPC-3ANTI-IGF-1R Ab# A 51 (Ganitumab; SEQ ID 327 + SEQ ID 328)

Example 14 Anti-IGF1R+Anti-ErbB3 BPAs Inhibit Both IGF1 and IGF2Mediated Signaling

This example shows that anti-IGF1R+anti-ErbB3 BPAs inhibit signalinginduced by IGF1 and IGF2.

The results were obtained essentially as follows. 500,000 DU145 and MiaPaCa-2 cells per well are plated in 12-well plates overnight in 10%serum. On day 2 cells are serum starved overnight. On day 3 antibodypre-incubations are performed for 1 hr (250 nM P4-G1-M1.3 or P4-G1-C8)and growth factors (IGF1 or IGF2 at 100 ng/ml) are added for 15 minutesprior to lysis. All cells are washed with PBS and lysed in 100 ul ofMPer buffer supplemented with protease and phosphatase inhibitors. Priorto running the samples on 4-12% Bis-Tris gels, loading buffer containingb-Mercaptoethanol (b-ME) is added and lysates are boiled for 5 minutesat 95° C. Gels are run at 150 volts constant for approximately 90minutes and transferred to nitrocellulose membranes using the iBlot(Invitrogen) transfer system's 8 minute transfer program. Membranes areblocked in Odyssey Blocking Buffer (Licor Biosciences) for 1 hour atroom temperature, and then incubated with primary antibodies overnightat 4 degrees C. in 5% BSA in TBS-T. Antibodies used are pAkt, pIGF1R,Beta Actin (all from Cell Signaling Technologies). B-Actin was used at1:5,000, Phospho-Akt at 1:2,000, and all others at 1:1,000. Thefollowing day membranes are washed 3×5 minutes each with TBS-T and thenincubated with anti-Rabbit IgG-DyLight800 (Cell Signaling) at 1:15,000in 5% milk in TBS-T for 1 hour at room temperature. Membranes are thenwashed 3×5 minutes each with TBS-T and scanned using the Licor Odysseysystem (Licor Biosciences). Integrated intensities are calculated andnormalized to Beta-Actin levels.

The results, (FIG. 45), show that BPAs P4-G1-M1.3 and P4-G1-C8 inhibitAKT phosphorylation induced by either IGF1 or IGF2.

Example 15 Anti-IGF1R+Anti-ErbB3 BPAs Partially Inhibit InsulinSignaling

This example shows that anti-IGF1R+anti-ErbB3 BPAs partially inhibitinsulin signaling in DU145 cells.

The results were obtained essentially as follows. 500,000 DU145 cellsper well are plated in 12-well plates overnight in 10% serum. On day 2cells are serum starved overnight. On day 3 Ab pre-incubations areperformed for 1 hr (500 nM P4-G1-M1.3) and growth factors (IGF1 at 100ng/ml or Insulin at 5 ug/ml) are added for 15 minutes prior to lysis.Lysates and Western blots are prepared as described in Example 14.

The results, (FIG. 46), indicate that BPA P4-G1-M1.3 partially inhibitssignal transduction induced by insulin, as measured by pAKT levels.

Example 16 Anti-IGF1R+Anti-ErbB3 BPAs Downregulate Total Receptor Levelsof ErbB3 and IGF1R

This Example shows that anti-IGF1R+anti-ErbB3 BPAs downregulate ErbB3and IGF1R levels on cells that are either induced by IGF1 and HRG or notinduced.

The results were obtained essentially as follows. 500,000 BxPC-3 cellsper well are plated in 12-well plates overnight in 10% serum. On day 2cells are serum starved overnight. On day 3 antibody pre-incubations areperformed for 6 hr (250 nM M7-G1-M78 or P4-G1-C8). To half of thesamples growth factors (IGF1 at 100 ng/ml and HRG at 30 ng/ml) are alsoadded for 15 minutes prior to lysis. Lysates and Western blots areprepared as described in Example 14. IGF1R, ErbB3 and pErbB3 are alsofrom Cell Signaling Technologies.

The results, (FIG. 47), indicate that total levels (phosphorylated andnon-phosphorylated) of ErbB3 and IGF1R are reduced by BPAs M7-G1-M78 andP4-G1-C8.

Example 17 Anti-IGF1R+Anti-ErbB3 BPAs Display Stability in Human, Mouseand Monkey Serum

This Example shows that anti-IGF1R+anti-ErbB3 BPAs are stable in human,mouse and monkey serum.

The results were obtained essentially as follows. PBAs are incubated ineither pooled human serum (Innovative Research), mouse serum (Sigma) orCynomolgous monkey serum (Innovative Research) at a final concentrationof 2.5 uM for either 0 days or 5 days at 37° C. The samples are thenassayed using a colorimetric ELISA binding assay. 96-well Reacti-bindplates (Pierce, Fisher cat. No. PI-15041) are coated with 50 ul of theprotein corresponding to the antibody's scFv (either ErbB3-His orIGF1R-His (R&D Systems, cat. No. 348-RB and 305-GR, respectively) at 2ug/ml in PBS) and incubated overnight at 4° C. The next day plates arewashed with PBS+0.05% Tween-20 (PBS-T) and blocked for 1 hr. at roomtemperature with 100 ul of Protein-Free Blocking Buffer (Pierce). Platesare washed with PBS-T and 50 ul of each BPA is added in duplicate.Concentrations start at 500 nM (1:5 dilution of 2.5 uM bsAb in serum) inPBS-T and include ten additional two-fold dilutions (in PBS-T+20% serum)and one blank (PBS-T+Serum only). Plates are incubated at roomtemperature for two hours and then washed with PBS-T. 50 ul ofanti-Fc-HRP (Jackson Labs) is added at 1:40,000 in PBS-T, and plates areincubated in the dark for 1 hr. at room temperature. Plates are againwashed with PBS-T and 100 ul of TMB substrate (Thermo Scientific, TMBand peroxide solution mixed 1:1) is added. The plates are incubated for5-15 minutes at room temperature until a blue color develops, and thereaction is stopped with 100 ul of STOP solution (Cell SignalingTechnology). The absorbance is read at 450 nm on a PerkinElmer Envisionplate reader, and binding curves are generated with GraphPad Prism.

The results, (FIG. 48), show that BPAs M7-G1-M78, P4-G1-M1.3, andP4-G1-C8 are stable for at least 5 days in mouse and cyno serum and thatP4-G1-M1.3 is stable for at least 6 days in human serum.

Example 18 Anti-IGF1R+Anti-ErbB3 BPAs Display Cross-Reactivity withHuman, Mouse, Rat and Monkey IGF1R and ErbB3

This example shows that anti-IGF1R+anti-ErbB3 BPAs are cross-reactivewith human, mouse, rat and monkey IGF1R and ErbB3.

Results were obtained essentially as follows. 96-well Reacti-bind®plates (Pierce, Fisher cat. No. PI-I5041) are coated with 50 ul ofspecies specific ErbB3-His or IGF-1R-His (R&D Systems, cat. No. 348-RBand 305-GR, respectively) at 2 ug/ml in PBS and incubated overnight at4° C. The next day plates are washed with PBS+0.05% Tween-20 (PBS-T) andblocked for 1 hr. at room temperature with 100 ul of Protein-FreeBlocking Buffer (Pierce). Plates are washed with PBS-T and 50 ul of eachPBA is added in duplicate. Concentrations start at 500 nM (in PBS-T) andincluded ten additional two-fold dilutions and one blank (PBS-T only).Plates are incubated at room temperature for two hours and then washedwith PBS-T. 50 ul of anti-Fc-HRP (Jackson Labs) is added at 1:40,000 inPBS-T, and plates are incubated in the dark for 1 hr. at roomtemperature. Plates are again washed with PBS-T and 100 ul of TMBsubstrate (Thermo Scientific, TMB and peroxide solution mixed 1:1) isadded. The plates are incubated for 5-15 minutes at room temperatureuntil a blue color develops, and the reaction is stopped with 100 ul ofSTOP solution (Cell Signaling Technology). The absorbance is read at 450nm on a PerkinElmer Envision plate reader, and binding curves aregenerated using GraphPad Prism.

The results, (FIG. 49), show that the BPAs P4-G1-C8, P4-G1-M1.3 andM7-G1-M78 bind efficiently to human, mouse, rat and monkey IGF1R andErbB3.

Example 19 Anti-IGF1R+Anti-ErbB3 BPAs Block IGF1 and IGF2 Binding totheir Receptor IGF1R

This Example shows that anti-IGF1R+anti-ErbB3 BPAs block the binding ofboth IGF1 and IGF2 to IGF1R.

The results were obtained essentially as follows. ELISA plates arecoated with IGF1R-His and blocked as described in Example 18. Followingthe blocking step, plates are washed and incubated with IGF1 (100 ng/ml)or IGF2 (100 ng/ml) (EMD Chemicals) for 1 hour at room temperature.Plates are washed with PBS-T, and 100 ul of each Ab is added induplicate. Concentrations start at 500 nM (in PBS-T) and include tenadditional two-fold dilutions and one blank (PBS-T only). Plates areincubated at room temperature for 1 hour and then washed with PBS-T. 50ul of Rabbit anti-Human IGF1 (Thermo Scientific) or Rabbit anti-HumanIGF2 (Abcam) are added at 1:1,000 in PBS-T for 1 hr. at roomtemperature. Plates are again washed and incubated in Anti-Rabbit HRP(Cell Signaling) at 1:1,000 in PBS-T for 1 hour at room temperature.Plates are developed, read and analyzed as described above.

The results, (FIG. 50), indicate that P4-G1-M1.3 inhibited the bindingof both IGF1 and IGF2 binding to IGF1R.

Example 20 Anti-IGF1R+Anti-ErbB3 BPAs Display Dose-Dependent andDifferent Half Lives in Mice and Long Half Lives in Cynomolgus Monkeys

This Example provides pharmacokinetic properties ofanti-IGF1R+anti-ErbB3 BPAs in mice and Cynomolgus monkeys.

The results were obtained essentially as follows. Dosing and collectionof samples: Mice are dosed by IV bolus with P4-G1-M1.3 or M7-G1-M78 ateither 100 ug/mouse or 500 ug/mouse and bleeds are taken at 0.25, 1, 4,8, 24, 48, 72, 96 and 168 hours. Four mice are bled per timepoint. IVinfusion of Cynomolgus Monkeys (WIL Research Laboratories) is performedwith P4-C8 and P4-M1.3. Two monkeys in each group are dosed at either 5mg/kg or 25 mg/kg and bled at 0.08, 1, 4, 8, 24, 48, 72, 96 and 168hours. ELISA binding assay and modeling analysis: Reacti-bind® 96-wellplates (Pierce, Fisher cat. No. PI-15041) are coated with 50 ul of IGF1R(No Tag) at 2 ug/ml in PBS and incubated overnight at 4° C. Plates arewashed with PBS-0.05% Tween-20 (PBS-T) and blocked for 1 hr at roomtemperature with 100 ul of Pierce Protein-Free Blocking Buffer. Platesare again washed with PBS-T. 100 ul of samples and standards are addedto plates and incubate for 2 hrs at room temperature. For standardcurves the antibodies are diluted to 12 ug/ml in PBS-T, then 10additional 3-fold dilutions with the final well blank. Serum samples arediluted at 1:50 in PBS-T with 10 additional 3-fold dilutions and a finalwell blank. Plates are washed with PBS-T and 100 ul of ErbB3-His addedat 1 ug/ml in PBS-T for 1 hr at room temperature. Plates are washed and100 ul anti-His-HRP (Abcam) is added at 1:10,000 in PBS-T and incubated(covered) for 1 hr. at room temperature. Plates are again washed withPBS-T and 100 ul of TMB substrate (Thermo Scientific, TMB and peroxidesolution mixed 1:1) was added. The plates are incubated for 5-15 minutesat room temperature until a blue color develops, and the reaction isstopped with 100 ul of STOP solution (Cell Signaling Technology). Theabsorbance is read at 450 nm on a PerkinElmer Envision plate reader, andthe data analysis is performed using MATLAB(Mathworks—www.mathworks.com) and WinNonLin(Pharsight—www.pharsight.com) according to standard protocols.

The results in mice, (Table 30), indicate that the BPAs M7-G1-M78 andP4-G1-M1.3 have a half life in mice that ranges from 3.33 to 41.90 hourson average, depending on the BPA and on the concentration of BPAadministered to the mouse. The results in Cynomolgus monkey, (Table 31),indicate that the half live of P4-G1-C8 and P4-G1-M1.3 is 51 and 61hours, respectively, for 5 mg/kg, and 115 and 78 hours, respectively,for 25 mg/kg of PBA. Thus, PBAs having the orientationanti-IGF1R-anti-ErbB3 (i.e., in which the anti-IGF1R portion is a fulllength Ab and the anti-ErbB3 portion is made up of two scFvs) are morestable than a PBA having the opposite conformation (i.e., in which theanti-ErbB3 is a full length Ab and the anti-IGF1R portion is made up oftwo scFvs).

TABLE 30 Half-life (in hours) of BPAs in mice Dose T ½ (hr) Molecule(ug/mouse) Mean 95% Conf Interval M7-G1-M78 100 3.33 2.43 5.32 500 11.169.40 13.75 P4-G1-M1.3 100 10.91 8.24 16.11 500 41.90 28.79 76.98

TABLE 31 Half-life (in hours) of BPAs in Cynomolgus monkeys MATLABWinNonLin Dose Terminal Terminal Molecule (mg/kg) T½ (hrs) 95% CI T/12(hrs) Rsq P4-C8 5 51 39-73  46.8529 0.9998 25 115 83-189 93.5036 0.959P4-M1.3 5 61 39-140 62.1732 0.9901 25 78 57-123 80.8655 0.9934

Example 21 P4-G1-M1.3 Displays In Vitro Inhibition of Ligand-InducedReceptor Activation and Akt/mTOR/ERK Signaling Over Time

This Example shows that P4-G1-M1.3 inhibits ligand-inducedphosphorylation of IGF-1R and ErbB3 as well as the downstream proteinsAkt, Erk, mTOR and S6 in BxPC-3 cultured cells over time.

Methods

BxPC-3 cells are maintained in RPMI-1640 media supplemented with 10%fetal bovine serum, Penicillin/Streptomycin and L-glutamine. 3.5×10⁴cells are plated in complete medium in 96-well tissue culture plates.The following day, complete medium is replaced with serum-free medium,and cells are incubated overnight at 37° C. Cells are either pretreatedfor 1 hour with 1 μM of P4-G1-M1.3 or kept in serum-free medium withoutinhibitor, and then all cells are stimulated for 5, 15, 30, 60 or 120minutes with 100 ng/ml IGF1 (Calbiochem) and 70 ng/ml HRG (R&D Systems).Cells are washed with PBS and lysed in MPer buffer supplemented withprotease and phosphatase inhibitors.

ELISAs for phospho-IGF1R (pIGF1R) phospho-ErbB3 (pErbB3) and phospho-AKT(pAKT) are performed as described in Example 4, above. ELISAs forphospho-ERK (pERK, Cell Signaling Technology catalog #7246), phospho-S6(pS6, R&D Systems catalog #DYC3918) and phospho-mTOR (pmTOR, R&D Systemscatalog #DYC1665) are performed according the manufacturer'sinstructions. Resulting concentrations of each phosphorylated proteinare normalized to levels of total protein, determined using the BCAmethod.

The results, (FIG. 51), indicate that P4-G1-M1.3 is able tosignificantly block ligand-induced production of Phospho-IGF-1R (51A),Phospho-ErbB3 (51B), Phospho-Akt (51C), Phospho-ERK (p44/p42; 51D),Phospho-mTOR (Ser2448, 51E), and Phospho-S6 (Ser235/236; 51F) in thepresence of IGF-1 and HRG.

Example 22 Anti-IGF1R+Anti-ErbB3 PBAs Block Signaling Mediated byIGF1R-Insulin Receptor Heterodimers

This Example shows that the inhibition of insulin and IGF2 signaling byanti-IGF1R+anti-ErbB3 PBAs is mediated by IGF1R-insulin receptorheterodimers.

The results were obtained essentially as follows. 500,000 BxPC-3 andA673 cells per well are plated in 12-well plates overnight in 10% serum.On day 2 cells are serum starved overnight. On day 3 antibodypre-incubations are performed for 1 hr (500 nM P4-G1-M1.3) and growthfactors (IGF1, IGF2 at 100 ng/ml or Insulin at 5 ug/ml) are added for 15minutes prior to lysis.

The results, (FIG. 52), indicate that P4-G1-M1.3 is able tosignificantly block IGF2 and insulin signaling in A673 cells, whichexpress high levels of Insulin Receptor (IR). This level of inhibitionwas not seen in BxPC-3 cells which express low IR levels.

Example 23 PBA P4-G1-M1.3 Displays Superior Reductions of mTORActivation and mTOR Protein Levels Relative to an Anti-IGF-1R mAb

This Example shows that P4-G1-M1.3 displays superior control of mTORactivation relative to an anti-IGF-1R mAb in end-of-study BxPC-3 tumors.

The profiling of tumors from a BxPC-3 xenograft study was conductedessentially as follows. Mice with human tumor cell line xenografts wereprepared and various treatments administered essentially as describedabove in Example 3D. Five end-of-study tumors were harvested from thePBS control group, the highest dose group of P4-G1-M1.3 (500 ug/mouseq3d), and the highest dose group of anti-IGF1R Ab# A (ganitumab; SEQ IDNO:327+SEQ ID NO:328) (368 ug/mouse q3d), and prepared essentially asfollows. Lysates are generated by tissue pulverization and lysis in TER1buffer (Invitrogen). Total protein is quantified by the BCA method, andequivalent total protein is run on 4-12% SDS-PAGE gels. Gels aretransferred to nitrocellulose using standard methods. Western blottingis performed using: anti-mTOR and anti-phospho-mTOR (Ser2448) primaryantibodies (all from Cell Signaling Technology). Secondary antibody usedis anti-Rabbit IgG-DyeLight800 (Cell Signaling Technology). Blots aredeveloped using the Li-Cor Odyssey system. Normalization to β-Actin isperformed by dividing the intensity of the target band by its associatedβ-Actin control band.

The results, (FIGS. 53A and B and Table 32), indicate that mTOR andphospho-mTOR(Ser2448) levels are significantly lower in tumors from micetreated with the PBA P4-G1-M1.3, relative to mice treated with theanti-IGF-1R Ab# A.

TABLE 32 P4-G1-M1.3 displays superior control of mTOR activity, relativeto anti-IGF-1R Ab# A in end-of-study BxPC-3 tumors Levels of Levels ofmTOR phospho-mTOR relative to relative to Samples buffer control buffercontrol Buffer control 1.0 1.0 P4-G1-M1.3 2.4 1.1 ANTI-IGF-1R 10.5 2.3Ab#A (ganitumab; SEQ ID NO: 327 + SEQ ID NO: 328)

Example 24 P4-G1-M1.3 Downregulates Receptors and Inhibits PI3K/Akt/mTORSignaling in Caki-1 and BxPC-3 Xenograft Models

This Example shows that P4-G1-M1.3 downregulates receptors and inhibitsPI3K/Akt/mTOR signaling in Caki-1 human renal clear cell carcinoma andBxPC-3 human pancreatic adenocarcinoma xenograft models in end-of-studytumors.

The profiling of tumors from a Caki-1 xenograftstudy was conductedessentially as follows. Mice with human tumor cell line xenografts wereprepared and various treatments administered essentially as describedabove in Example 3D. For the Caki-1 xenograft study, 3 groups wereestablished, each containing 5 mice. These included control, P4-G1-M1.3(600 μg), and anti-IGF-1R Ab# A (291 μg dose 1, 320 μg dose 2).Antibodies were dosed twice, IP, at a three day interval. Everolimus wasdosed PO, qd. Tumors were harvested 24 hours after the second antibodydose.

The profiling of tumors from a BxPC-3 xenograftstudy was conductedessentially as described in Example 23.

Tumors were initially weighed and pulverized in a CryoPrep tissuepulverizer (Model CP-02, Covaris). Tissue Extraction Reagent 1 (TER1,Life Technologies™) containing protease and phosphatase inhibitors wasadded to the tumor at a ratio of 1 ml TER1 per 100 mg of tissue. Sampleswere incubated on ice for 30 minutes to solubilize tissue and putthrough a QIAshredder™ column (Qiagen) according to the manufacturersprotocol. A BCA assay (Pierce) was performed to determine proteinconcentration according to the manufacturer's protocol.

Samples were analyzed by western blot. Loading buffer containing13-Mercaptoethanol (β-ME) was added and lysates were boiled for 5minutes at 95° C. Approximately 40 μg of protein and two ladders(Invitrogen) were run on each well of an 18-well gel (BioRad). Gels wererun at 150 volts constant for approximately 90 minutes and transferredto nitrocellulose membranes using the iBlot® (Invitrogen) transfersystem's 8 minute transfer program. Membranes were blocked in Odyssey®Blocking Buffer (Licor® Biosciences) for 1 hour at room temperature, andthen incubated with primary antibodies overnight at 4° C. in 5% BSA inTBS-T. All antibodies were purchased from Cell Signaling and used at therecommended dilution. The following day membranes were washed 3×5minutes each with TBS-T and then incubated with anti-Rabbit IgG-DyLight®800 (Cell Signaling) or anti-Rabbit IRDye® 800 (Licor® Biosciences) at1:10,000-15,000 in 5% milk in TBS-T for 1 hour at room temperature.Membranes were then washed 3×5 minutes each with TBS-T and scanned usingthe Licor® Odyssey® system (Licor® Biosciences). Band intensities werequantified using Image Studio 2.0 and normalized to β-Actin levels. InCaki-1 xenografts control tumors were compared to those treated withP4-G1-M1.3, Anti-IGF-1R Ab#A+Anti-ErbB3 Ab# A, or everolimus.

The results of the Caki-1 profiling study, (FIGS. 54 A-F), indicate thatlevels of IGF-1R, insulin receptor, ErbB3, EGFR, pAKT(Ser473 or Thr308),pFox01(Thr24)/Fox03a(Thr32) and phospho-mTOR(Ser2448 or Ser2481) are allsimilar to levels in control mice or lower in tumors from mice treatedwith P4-G1-M1.3, relative to mice treated with the Anti-IGF-1R Ab#A+Anti-ErbB3 Ab# A combination or the mTOR inhibitor everolimus.

The results of the BxPC-3 profiling study, (FIGS. 55 A-C), indicate thatIGF-1R, ErbB3, pEGFR, pmTOR(S2448), and pS6(S235/236) are all lower intumors from mice treated with P4-G1-M1.3, relative to mice treated withthe Anti-IGF-1R Ab#A or PBS alone.

Example 25 P4-G1-M1.3 Blocks IGF-1 and IGF-2 Binding to Receptors

This Example shows by means of an ELISA assay that P4-G1-M1.3effectively blocks the binding of IGF-1 and IGF-2 to IGF-1R.

96-well Reacti-bind® plates (Pierce) were coated with 50 μl ofIGF-1R-His (R&D Systems cat. No. 305-GR; 2 μg/ml in PBS) and incubatedovernight at 4° C. The next day plates were washed with PBS+0.05%Tween-20 (PBS-T) and blocked for 1 hr. at room temperature with 100 μlof Protein-Free Blocking Buffer (Pierce). Plates were washed with PBS-Tand 100 μl of P4-G1-M1.3 was added in duplicate. Antibody concentrationstarted at 500 nM (in PBS-T) and included ten additional two-folddilutions and one blank (PBS-T only). Plates were incubated at roomtemperature for two hours and then washed with PBS-T. 100 μl of eitherIGF-1 or IGF-2 (EMD Chemicals) was added at 100 ng/ml and incubated atroom temperature for one hour. Following washing, 100 μl of eitherRabbit-Anti-IGF-1 or Rabbit-Anti-IGF-2 (both Abcam, 5 μg/ml) were addedto the plates and incubated for one hour at room temperature. Plateswere then washed and incubated with 100 μl of Anti-Rabbit-HRP (CellSignaling) for 1 hour at room temperature, washed again and 100 μl ofTMB substrate (Cell Signaling) was added. The plates were incubated for5-15 minutes at room temperature until a blue color developed, and thereaction was stopped with 100 μl of STOP solution (Cell SignalingTechnology). The absorbance was read at 450 nm on a PerkinElmer Envisionplate reader, and binding curves were generated using GraphPad Prism®.

The results of the ELISA assay, (FIG. 56), show that P4-G1-M1.3 blocksboth IGF-1 and IGF-2 binding to IGF-1R in a dose-dependent manner.

Example 26 P4-G1-M1.3 and P4-G1-C8 in DU145, BxPC-3, SK-ES-1, and Caki-1Tumor Xenograft Models

For each of studies A-D below, cells were respsusended 1:1 withPBS:Growth factor-reduced Matrigel® and injected subcutaneously intoNu/Nu mice. Tumors were allowed to develop for 8 days. Antibodies wereinjected intraperitoneally every 3 days (q3d) at the indicateddoses/mouse. Tumor lengths and widths were measured twice a weekmanually by caliper, and tumor volume calculated using the followingformula: π/6(L×W²). Each arm of the study contained 10 animals.

A. P4-G1-C8 and P4-G1-M1.3 Suppress Tumor Growth of DU145 ProstateCancer Cells In Vivo.

For this DU145 xenograft study, 8×10⁶ DU145 cells were prepared and usedas described above.

The results (FIG. 57A), show that both P4-G1-C8 and P4-G1-M.3 suppressthe growth of prostate cancer cells in vivo.

B. P4-G1-C8 and P4-G1-M1.3 Suppress Tumor Growth of BxPC-3 PancreaticCancer Cells In Vivo Better than an Anti-IGF-1R IgG.

For this aft study, 5×10⁶ BxPC-3 cells were prepared and used asdescribed above.

The results (FIG. 57B), show that P4-G1-C8 and P4-G1-M1.3 suppress tumorgrowth of BxPC-3 pancreatic cancer cells in vivo and that both aresuperior to anti-IGF-1R Ab# A in inhibiting tumor cell growth.

C. P4-G1-M1.3 Suppresses Tumor Growth of SK-ES-1 Ewing's Sarcoma CancerCells In Vivo

For this xenograft study, 10×10⁶ SK-ES-1 cells were prepared and used asdescribed above.

The results (FIG. 57C), show that P4-G1-M1.3 suppresses tumor cellgrowth in a dose-dependent manner.

D. P4-G1-M1.3 Suppresses Tumor Growth of Caki-1 Renal Cell CarcinomaCancer Cells In Vivo and Displays Superior Suppression Compared to theCombination of an Anti-IGF-1R IgG and an Anti-ErbB3 IgG.

For this study, 8×10⁶ Caki-1 cells were prepared and used as describedabove.

The results (FIG. 57D), show that P4-G1-M1.3 suppresses tumor cellgrowth in a dose-dependent manner and is more effective at inhibitingtumor cell growth that a combination of anti-IGF-1R Ab# A (ganitumab;SEQ ID NO:327+SEQ ID NO:328) and anti-ErbB3 Ab# A (SEQ ID NO:336+SEQ IDNO:337) antibody, whether these antibodies are given at an equalexposure or at equimolar dosing.

Example 27 Computational Analysis of PK Data to Design PD/EfficacyStudies in Mice

This Example describes the computational methodology used to fitmathematical modeling to experimental data to estimate thepharmacokinetics (PK) parameters of M1.3-G1-P4.

Fitting Mathematical Modeling to PK Data

The PK parameters of M1.3-G1-P4 were inferred via implementation of theintravenous (IV) bolus target mediated drug disposition (TMDD) model.The IV TMDD is a 2-compartment PK model structured as a set of 4 coupleddifferential equations, as follows:

$\begin{matrix}{\frac{\lbrack{Dc}\rbrack}{t} = {{- {C_{ld}\lbrack{Dc}\rbrack}} + {{C_{ld}\lbrack{Dp}\rbrack}\frac{Vp}{Vc}} - {C_{l}\lbrack{Dc}\rbrack} - {{k_{on}\lbrack R\rbrack}\lbrack{Dc}\rbrack} + {k_{off}\left\lbrack {D:R} \right\rbrack}}} & \left( {1\; A} \right) \\{\frac{\lbrack{Dp}\rbrack}{t} = {{{C_{ld}\lbrack{Dc}\rbrack}\frac{Vc}{Vp}} - {C_{ld}\lbrack{Dp}\rbrack}}} & \left( {1\; B} \right) \\{\frac{\lbrack R\rbrack}{t} = {k_{in} - {k_{out}\lbrack R\rbrack} - {{k_{on}\lbrack R\rbrack}\lbrack{Dc}\rbrack} + {k_{off}\left\lbrack {D:R} \right\rbrack}}} & \left( {1\; C} \right\rbrack \\{\frac{\left\lbrack {D:R} \right\rbrack}{t} = {{{k_{on}\lbrack R\rbrack}\lbrack{Dc}\rbrack} - {k_{off}\left\lbrack {D:R} \right\rbrack} - {k_{el}\left\lbrack {D:R} \right\rbrack}}} & \left( {1\; D} \right)\end{matrix}$

In equations 1A-1D, Dc and Dp are the concentrations of the drug in thecentral and peripheral compartments, Vc and Vp are the volumes of thecentral and peripheral compartments, whereas R and D:R denote theconcentrations of the free target receptor and drug-receptor complex inthe central compartment. Moreover, C_(ld), C_(l), k_(in), k_(out),k_(on), k_(off) and k_(el) respectively represent the rate constants oftransport across compartments, drug clearance from the centralcompartment, receptor synthesis, receptor degradation, drug-receptorassociation, drug-receptor dissociation, and drug-receptor clearancefrom the central compartment. The IV TMDD model describes the dynamicsprocesses of drug binding to the target receptor and drug clearance inthe central compartment as well as the process of drug transport fromthe central to the peripheral compartment when the drug of interest isdirectly injected in the central compartment (blood). Fitting TMDD modelto experimental data obtained from mice blood enables estimating the PKproperties of M1.3-G1-P4.

FIG. 58 shows the fitting of TMDD model to experimental data (solidline=fit of 500 μg/mouse dose given i.v., dotted line=fit of 100μg/mouse dose given i.v.). The M1.3-G1-P4 PK parameters are listed inTable 24 below.

TABLE 24 PK parameters of M1.3-G1-P4 inferred from fitting of IV TMDDmodeling to PK data obtained from mice blood C_(ld) C_(l) k_(in) k_(out)k_(on) k_(off) k_(el) Vc Vt (h⁻¹) (h⁻¹) (ng mL⁻¹ h⁻¹) (h⁻¹) (ng⁻¹ k⁻¹)(h⁻¹) (k⁻¹) mL mL M1.3-G1-P4 0.203 0.030 1020.44 0.0415 0.006 0.010.0001 1.386 51.745

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain andimplement using no more than routine experimentation, many equivalentsof the specific embodiments described herein. Such equivalents areintended to be encompassed by the following claims. Any combinations ofthe embodiments disclosed in the dependent claims are contemplated to bewithin the scope of the disclosure.

INCORPORATION BY REFERENCE

The disclosure of each and every U.S. and foreign patent and pendingpatent application and publication referred to herein is specificallyincorporated by reference herein in its entirety.

1-194. (canceled)
 195. A polyvalent bispecific antibody (PBA), whichantibody is a protein comprising two pairs of polypeptide chains, eachpair of said two pairs comprising a heavy chain joined to a light chainby at least one heavy-light chain bond; wherein each pair comprises atleast one anti-IGF-1R binding site and at least one anti-ErB3 bindingsite; and each pair comprises a first binding site that comprises anN-terminal portion of the heavy chain of the PBA and an N-terminalportion of the light chain of the PBA, and a second binding site that isa C-terminal scFv that is entirely comprised by the heavy chain of thePBA, said C-terminal scFv containing a heavy chain variable regionjoined to a light chain variable region by an scFv linker; and theanti-IGF-1R binding site is linked to the anti-ErbB3 binding sitethrough a heavy chain immunoglobulin (HC Ig) constant region comprisedby the heavy chain of the PBA, and the two pairs are conjoined by atleast one bond between the HC Ig constant regions of each pair, and (i)the anti-IGF-1R binding site comprises a heavy chain variable (VH)domain comprising a set of three VH Complementarity Determining Regions(CDRs) comprising either (a) VHCDR1 (amino acid numbers 26-35), VHCDR2(amino acid numbers 51-66), and VHCDR3 (amino acid numbers 99-111), of aheavy chain having an amino acid sequence comprising the amino acidsequence of a SEQ ID NO selected from the group consisting of SEQ IDNO:1, SEQ ID NOs:8-31 and SEQ ID NOs:384-385; or (b) a set of three VHComplementarity Determining Regions (CDRs) comprising VHCDR1 comprisingSEQ ID NO:302, VHCDR2 comprising SEQ ID NO:303 and VHCDR3 comprising SEQID NO:304; and a light chain variable (VL) domain comprising a set ofthree VLCDRs comprising either (c) VLCDR1 (amino acid numbers 24-34),VLCDR2 (amino acid numbers 50-56) and VLCDR3 (amino acid numbers 89-97)of a light chain having an amino acid sequence comprising the amino acidsequence of a SEQ ID NO selected from the group consisting of SEQ IDNOs:2-3, SEQ ID NOs:32-133, and SEQ ID NOs:386-387; or (d) a set ofthree VLCDRs comprising VLCDR1 comprising SEQ ID NO:305, VLCDR2comprising SEQ ID NO:306 and VLCDR3 comprising SEQ ID NO:307 or SEQ IDNO:308, and each CDR further comprising an amino terminus and a carboxyterminus, wherein the CDRs of each set of CDRs are arranged in thecorresponding heavy or light chain in a linear amino to carboxy order ofCDR1, CDR2 and CDR3, and (ii) the anti-ErbB3 binding site comprises aheavy chain variable (VH) domain comprising a set of three VH CDRscomprising either (e) VHCDR1 (amino acid numbers 26-35), VHCDR2 (aminoacid numbers 51-66) and VHCDR3 (amino acid numbers 99-111) of a heavychain having an amino acid sequence comprising the amino acid sequenceof a SEQ ID NO selected from the group consisting of SEQ ID NOs:4-5, SEQID NOs: 134-165, and SEQ ID NO:388, or (f) a set of three VH CDRscomprising VHCDR1 comprising SEQ ID NO:309, VHCDR2 comprising SEQ IDNO:310 and VHCDR3 comprising SEQ ID NO:311, and a light chain variable(VL) domain comprising a set of three VLCDRs comprising either (g)VLCDR1 (amino acid numbers 23-33), VLCDR2 (amino acid numbers 49-55) andVLCDR3 (amino acid numbers 88-98), of a light chain having an amino acidsequence comprising the amino acid sequence of a SEQ ID NO selected fromthe group consisting of SEQ ID NOs:6-7 and SEQ ID NOs: 166-200; or (h) alight chain variable (VL) domain comprising a set of three VLCDRscomprising VLCDR1 comprising SEQ ID NO:312, VLCDR2 comprising SEQ IDNO:313 and VLCDR3 comprising SEQ ID NO:314 or SEQ ID NO:315; and eachCDR further comprising an amino terminus and a carboxy terminus, whereinthe CDRs of each set of CDRs are arranged in the antibody in a linearamino to carboxy order of CDR1, CDR2 and CDR3, and (iii) wherein the PBAdoes not comprise both a) an anti-IGF-1R module comprising a light chaincomprising SEQ ID NO:35 and a heavy chain comprising SEQ ID NO:11 and b)an anti-ErbB3 module comprising a light chain comprising SEQ ID NO:175and a heavy chain comprising SEQ ID NO:145.
 196. A polyvalent bispecificantibody that is: a. an SF-G1-P1 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:212; and two light chains, each comprising a lightchain sequence of SEQ ID NO:202, or; b. an SF-G1-M1.3 polyvalentbispecific antibody comprising: two heavy chains, each comprising aheavy chain amino acid sequence of SEQ ID NO:214; and two light chains,each comprising a light chain amino acid sequence of SEQ ID NO:202, or;c. an SF-G1-M27 polyvalent bispecific antibody comprising: two heavychains, each comprising a heavy chain amino acid sequence of SEQ IDNO:216; and two light chains, each comprising a light chain amino acidsequence of SEQ ID NO:202, or; d. an SF-G1-P6 polyvalent bispecificantibody comprising: two heavy chains, each comprising a heavy chainamino acid sequence of SEQ ID NO:218; and two light chains, eachcomprising a light chain amino acid sequence of SEQ ID NO:202, or; e. anSF-G1-B69 polyvalent bispecific antibody comprising: two heavy chains,each comprising a heavy chain amino acid sequence of SEQ ID NO:220; andtwo light chains, each comprising a light chain amino acid sequence ofSEQ ID NO:202, or; f. a P4-G1-P1 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:224; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:204, or; g. a P4-G1-M27polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:228; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:204, or; h. a P4-G1-P6 polyvalent bispecific antibody comprising:two heavy chains, each comprising a heavy chain amino acid sequence ofSEQ ID NO:230; and two light chains, each comprising a light chain aminoacid sequence of SEQ ID NO:204, or; i. a P4-G1-B69 polyvalent bispecificantibody comprising: two heavy chains, each comprising a heavy chainamino acid sequence of SEQ ID NO:232; and two light chains, eachcomprising a light chain amino acid sequence of SEQ ID NO:204, or; j. anM78-G1-C8 polyvalent bispecific antibody comprising: two heavy chains,each comprising a heavy chain amino acid sequence of SEQ ID NO:234; andtwo light chains, each comprising a light chain amino acid sequence ofSEQ ID NO:206, or; k. an M78-G1-P1 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:236; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:206, or; l. an M78-G1-M1.3polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:238; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:206, or; m. an M78-G1-M27 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:240; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:206, or; n. an M78-G1-P6polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:242; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:206, or; o. an M78-G1-B69 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:244; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:206, or; p. an M57-G1-C8polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:246; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:208, or; q. an M57-G1-P1 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:248; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:208, or; r. an M57-G1-M1.3polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:250; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:208, or; s. an M57-G1-M27 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:252; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:208, or; t. an M57-G1-P6polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:254; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:208, or; u. an M57-G1-B69 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:256; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:208, or; v. a P1-G1-P4 polyvalentbispecific antibody comprising: two heavy chains, each comprising aheavy chain amino acid sequence of SEQ ID NO:268; and two light chains,each comprising a light chain amino acid sequence of SEQ ID NO:258, or;w. a P1-G1-M57 polyvalent bispecific antibody comprising: two heavychains, each comprising a heavy chain amino acid sequence of SEQ IDNO:270; and two light chains, each comprising a light chain amino acidsequence of SEQ ID NO:258, or; x. A P1-G1-M78 polyvalent bispecificantibody comprising: two heavy chains, each comprising a heavy chainamino acid sequence of SEQ ID NO:272; and two light chains, eachcomprising a light chain amino acid sequence of SEQ ID NO:258, or; y. anM27-G1-P4 polyvalent bispecific antibody comprising: two heavy chains,each comprising a heavy chain amino acid sequence of SEQ ID NO:274; andtwo light chains, each comprising a light chain amino acid sequence ofSEQ ID NO:260, or; z. an M27-G1-M57 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:276; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:260, or; aa. an M27-G1-M78polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:278; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:260, or; bb. an M7-G1-P4 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:280; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:262, or; cc. an M7-G1-M57polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:282; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:262, or; dd. a B72-G1-P4 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:286; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:264, or; ee. a B72-G1-M57polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:288; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:264, or; ff. a B72-G1-M78 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:290; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:264, or; gg. a B60-G1-P4polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:292; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:266, or; hh. a B60-G1-M57 polyvalent bispecific antibodycomprising: two heavy chains, each comprising a heavy chain amino acidsequence of SEQ ID NO:294; and two light chains, each comprising a lightchain amino acid sequence of SEQ ID NO:266, or; ii. a B60-G1-M78polyvalent bispecific antibody comprising: two heavy chains, eachcomprising a heavy chain amino acid sequence of SEQ ID NO:296; and twolight chains, each comprising a light chain amino acid sequence of SEQID NO:266.
 197. An anti-IGF-1R monoclonal antibody a heavy chain and alight chain and comprising: jj. a first sequence comprising in amino tocarboxy order a VLCDR1 sequence, a VLCDR2 sequence and a VLCDR3 sequenceof SF kappa light chain as indicated by dotted underlining in FIG. 5A,SEQ ID NO:202, said antibody further comprising a second sequencecomprising in amino to carboxy order a VHCDR1 sequence, a VHCDR2sequence and a VHCDR3 sequence of SF heavy chain as indicated by thefirst three dotted underlined sequences respectively in FIG. 5A, SEQ IDNO:210, wherein the first sequence and the second sequence arenon-overlapping, or; kk. in amino to carboxy order a VLCDR1 sequence, aVLCDR2 sequence and a VLCDR3 sequence of P4 kappa light chain asindicated by dotted underlining in FIG. 5A, SEQ ID NO:204, said antibodyfurther comprising in amino to carboxy order a VHCDR1 sequence, a VHCDR2sequence and a VHCDR3 sequence of P4 heavy chain as indicated by thefirst three dotted underlined sequences respectively in FIG. 5A, SEQ IDNO:222, or; ll. in amino to carboxy order a VLCDR1 sequence, a VLCDR2sequence and a VLCDR3 sequence of M78 kappa light chain as indicated bydotted underlining in FIG. 5A, SEQ ID NO:206, said antibody furthercomprising in amino to carboxy order a VHCDR1 sequence, a VHCDR2sequence and a VHCDR3 sequence of M78 heavy chain as indicated by thefirst three dotted underlined sequences respectively in FIG. 5A, SEQ IDNO:234, or; mm. in amino to carboxy order a VLCDR1 sequence, a VLCDR2sequence and a VLCDR3 sequence of M57 kappa light chain as indicated bydotted underlining in FIG. 5A, SEQ ID NO:208, said antibody furthercomprising in amino to carboxy order a VHCDR1 sequence, a VHCDR2sequence and a VHCDR3 sequence of M57 heavy chain as indicated by thefirst three dotted underlined sequences respectively in FIG. 5A, SEQ IDNO:246, or nn. a VH domain comprising a set of three VH CDRs comprisingVHCDR1, VHCDR2, VHCDR3, and a VL domain comprising a set of three VLCDRs comprising VLCDR1, VLCDR2 and VLCDR3, said CDRs comprising thesequences of SEQ ID NOs: 302, 303, 304, 305, 306, and 307, respectively,and each CDR further comprising an amino terminus and a carboxyterminus, wherein the CDRs of each set of CDRs are arranged in theantibody in a linear amino to carboxy order of CDR1, CDR2, and CDR3 andthe CDRs comprise variable amino acids, which independently representany amino acid set forth at the corresponding position in FIG. 1 (VH) orFIG. 2 (VL), and wherein the antibody binds human IGF-1R and does notcomprise the SF module.
 198. An anti-ErbB3 monoclonal antibodycomprising a heavy chain and a light chain and comprising in amino tocarboxy order: oo. a VLCDR1 sequence, a VLCDR2 sequence and a VLCDR3sequence of P1 lambda light chain as indicated by dotted underlining inFIG. 5B, SEQ ID NO:258, said antibody further comprising in amino tocarboxy order a VHCDR1 sequence, a VHCDR2 sequence and a VHCDR3 sequenceof P1 heavy chain as indicated by the first three dotted underlinedsequences respectively in FIG. 5B, SEQ ID NO:268, or pp. a VLCDR1sequence, a VLCDR2 sequence and a VLCDR3 sequence of M27 lambda lightchain as indicated by dotted underlining in FIG. 5B, SEQ ID NO:260, saidantibody further comprising in amino to carboxy order a VHCDR1 sequence,a VHCDR2 sequence and a VHCDR3 sequence of M27 heavy chain as indicatedby the first three dotted underlined sequences respectively in FIG. 5B,SEQ ID NO:274, or; qq. a VLCDR1 sequence, a VLCDR2 sequence and a VLCDR3sequence of M7 lambda light chain as indicated by dotted underlining inFIG. 5B, SEQ ID NO:262, said antibody further comprising in amino tocarboxy order a VHCDR1 sequence, a VHCDR2 sequence and a VHCDR3 sequenceof M7 heavy chain as indicated by the first three dotted underlinedsequences respectively in FIG. 5B, SEQ ID NO:280, or; rr. a VLCDR1sequence, a VLCDR2 sequence and a VLCDR3 sequence of B72 lambda lightchain as indicated by dotted underlining in FIG. 5B, SEQ ID NO:264, saidantibody further comprising in amino to carboxy order a VHCDR1 sequence,a VHCDR2 sequence and a VHCDR3 sequence of B72 heavy chain as indicatedby the first three dotted underlined sequences respectively in FIG. 5B,SEQ ID NO:286, or; ss. a VLCDR1 sequence, a VLCDR2 sequence and a VLCDR3sequence of B60 lambda light chain as indicated by dotted underlining inFIG. 5B, SEQ ID NO:266, said antibody further comprising in amino tocarboxy order a VHCDR1 sequence, a VHCDR2 sequence and a VHCDR3 sequenceof B60 heavy chain as indicated by the first three dotted underlinedsequences respectively in FIG. 5B, SEQ ID NO:292, or; tt. a VH domaincomprising a set of three VH CDRs comprising VHCDR1, VHCDR2, VHCDR3, anda VL domain comprising a set of three VL CDRs comprising VLCDR1, VLCDR2and VLCDR3, said CDRs comprising the sequences of SEQ ID NOs: 309, 310,311, 312, 313, and 314, respectively, and each CDR further comprising anamino terminus and a carboxy terminus, wherein the CDRs of each set ofCDRs are arranged in the antibody in a linear amino to carboxy order ofCDR1, CDR2, and CDR3, wherein the CDRs comprise variable amino acids,which independently represent any amino acid set forth at thecorresponding position in FIG. 3 (VH) or FIG. 4 (VL), and the antibodydoes not comprise the C8 module.
 199. A method for treating a subjecthaving a cancer, said method comprising administering to the subject atherapeutically effective amount of the polyvalent bispecific antibodyof claim
 195. 200. A method for treating a subject having a cancer, saidmethod comprising administering to the subject a therapeuticallyeffective amount of the polyvalent bispecific antibody of claim 196.201. A method for treating a subject having a cancer, said methodcomprising administering to the subject a therapeutically effectiveamount of the monoclonal antibody of claim
 197. 202. A method fortreating a subject having a cancer, said method comprising administeringto the subject a therapeutically effective amount of the monoclonalantibody of claim
 198. 203. A nucleic acid molecule encoding the heavychain of the polyvalent bispecific antibody of claim
 195. 204. A nucleicacid molecule encoding the heavy chain of the polyvalent bispecificantibody of claim
 196. 205. A nucleic acid molecule encoding the heavychain of the monoclonal antibody of claim
 197. 206. A nucleic acidmolecule encoding the light chain of the monoclonal antibody of claim197.
 207. A nucleic acid molecule encoding the heavy chain of themonoclonal antibody of claim
 198. 208. A nucleic acid molecule encodingthe light chain of the monoclonal antibody of claim
 198. 209. Thepolyvalent bispecific antibody of claim 195 in a pharmaceuticallyacceptable carrier.
 210. The polyvalent bispecific antibody of claim 196in a pharmaceutically acceptable carrier.
 211. The monoclonal antibodyof claim 197 in a pharmaceutically acceptable carrier.
 212. Themonoclonal antibody of claim 198 in a pharmaceutically acceptablecarrier.